Molecular classification and biomarkers of clinical outcome in breast ductal carcinoma in situ: Analysis of TBCRC 038 and RAHBT cohorts.
Ductal carcinoma in situ (DCIS) is the most common precursor of invasive breast cancer (IBC), with variable propensity for progression. We perform multiscale, integrated molecular profiling of DCIS with clinical outcomes by analyzing 774 DCIS samples from 542 patients with 7.3 years median follow-up from the Translational Breast Cancer Research Consortium 038 study and the Resource of Archival Breast Tissue cohorts. We identify 812 genes associated with ipsilateral recurrence within 5 years from treatment and develop a classifier that predicts DCIS or IBC recurrence in both cohorts. Pathways associated with recurrence include proliferation, immune response, and metabolism. Distinct stromal expression patterns and immune cell compositions are identified. Our multiscale approach employed in situ methods to generate a spatially resolved atlas of breast precancers, where complementary modalities can be directly compared and correlated with conventional pathology findings, disease states, and clinical outcome.
View details for DOI 10.1016/j.ccell.2022.10.021
View details for PubMedID 36400020
Transition to invasive breast cancer is associated with progressive changes in the structure and composition of tumor stroma.
2022; 185 (2): 299-310.e18
Ductal carcinoma in situ (DCIS) is a pre-invasive lesion that is thought to be a precursor to invasive breast cancer (IBC). To understand the changes in the tumor microenvironment (TME) accompanying transition to IBC, we used multiplexed ion beam imaging by time of flight (MIBI-TOF) and a 37-plex antibody staining panel to interrogate 79 clinically annotated surgical resections using machine learning tools for cell segmentation, pixel-based clustering, and object morphometrics. Comparison of normal breast with patient-matched DCIS and IBC revealed coordinated transitions between four TME states that were delineated based on the location and function of myoepithelium, fibroblasts, and immune cells. Surprisingly, myoepithelial disruption was more advanced in DCIS patients that did not develop IBC, suggesting this process could be protective against recurrence. Taken together, this HTAN Breast PreCancer Atlas study offers insight into drivers of IBC relapse and emphasizes the importance of the TME in regulating these processes.
View details for DOI 10.1016/j.cell.2021.12.023
View details for PubMedID 35063072
Leptin-resistant Zucker rats with trinitrobenzene sulfonic acid colitis present a reduced inflammatory response but enhanced epithelial damage
AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY
2021; 321 (2): G157-G170
The role of leptin in the development of intestinal inflammation remains controversial, since proinflammatory and anti-inflammatory effects have been described. This study describes the effect of the absence of leptin signaling in intestinal inflammation. Experimental colitis was induced by intrarectal administration of trinitrobenzene sulfonic acid (TNBS) to lean and obese Zucker rats (n = 10). Effects on inflammation and mucosal barrier were studied. Bacterial translocation and LPS concentration were evaluated together with colonic permeability to 4-kDa FITC-dextran. Obese Zucker rats showed a lower intestinal myeloperoxidase and alkaline phosphatase activity, reduced alkaline phosphatase sensitivity to levamisole, and diminished colonic expression of Nos2, Tnf, and Il6, indicating attenuated intestinal inflammation, associated with attenuated STAT3, AKT, and ERK signaling in the colonic tissue. S100a8 and Cxcl1 mRNA levels were maintained, suggesting that in the absence of leptin signaling neutrophil activation rather than infiltration is hampered. Despite the lower inflammatory response, leptin resistance enhanced intestinal permeability, reflecting an increased epithelial damage. This was shown by augmented LPS presence in the portal vein of colitic obese Zucker rats, associated with induction of tissue nonspecific alkaline phosphatase, LPS-binding protein, and CD14 hepatic expression (involved in LPS handling). This was linked to decreased ZO-1 immunoreactivity in tight junctions and lower occludin expression. Our results indicate that obese Zucker rats present an attenuated inflammatory response to TNBS, but increased intestinal epithelial damage allowing the passage of bacterial antigens.NEW & NOTEWORTHY Obese Zucker rats, which are resistant to leptin, exhibit a diminished inflammatory response in the trinitrobenzenesulfonic acid (TNBS) model of colitis, suggesting leptin role is proinflammatory. At the same time, obese Zucker rats present a debilitated intestinal barrier function, with increased translocation of LPS. Zucker rats present a dual response in the TNBS model of rat colitis.
View details for DOI 10.1152/ajpgi.00367.2020
View details for Web of Science ID 000688299200007
View details for PubMedID 34132111
Immune cell topography predicts response to PD-1 blockade in cutaneous T cell lymphoma.
2021; 12 (1): 6726
Cutaneous T cell lymphomas (CTCL) are rare but aggressive cancers without effective treatments. While a subset of patients derive benefit from PD-1 blockade, there is a critically unmet need for predictive biomarkers of response. Herein, we perform CODEX multiplexed tissue imaging and RNA sequencing on 70 tumor regions from 14 advanced CTCL patients enrolled in a pembrolizumab clinical trial (NCT02243579). We find no differences in the frequencies of immune or tumor cells between responders and non-responders. Instead, we identify topographical differences between effector PD-1+ CD4+ T cells, tumor cells, and immunosuppressive Tregs, from which we derive a spatial biomarker, termed the SpatialScore, that correlates strongly with pembrolizumab response in CTCL. The SpatialScore coincides with differences in the functional immune state of the tumor microenvironment, T cell function, and tumor cell-specific chemokine recruitment and is validated using a simplified, clinically accessible tissue imaging platform. Collectively, these results provide a paradigm for investigating the spatial balance of effector and suppressive T cell activity and broadly leveraging this biomarker approach to inform the clinical use of immunotherapies.
View details for DOI 10.1038/s41467-021-26974-6
View details for PubMedID 34795254
Exogenous leptin reinforces intestinal barrier function and protects from colitis
2019; 147: 104356
Besides its function controlling energy expenditure and food intake, leptin is an important modulator of inflammatory responses. The role of leptin in intestinal inflammation remains controversial, since both pro-inflammatory and anti-inflammatory effects have been reported. This study was carried out to further understand leptin contribution in the inflamed intestinal mucosa. Exogenous PEG-leptin or saline solution was given to C57BL/6 mice for two weeks. After 1 week, acute colitis was induced to C57BL/6 mice using dextran sulfate sodium (DSS) in drinking water. The severity of colitis, inflammatory parameters and mucosal barrier function were evaluated. Overall our results indicate that colitis was less severe in mice receiving leptin, as shown by a decrease in rectal bleeding, epithelial damage and colon inflammatory markers, and improved diarrhea. Leptin-treated mice displayed an increase in the expression of tight junction proteins and proliferative expression markers in colon, indicating a reinforcement in the mucosal barrier function induced by leptin administration. PEG-leptin treatment conferred protection to mice in the DSS model of colitis by reinforcing mucosal barrier function.
View details for DOI 10.1016/j.phrs.2019.104356
View details for Web of Science ID 000487565500015
View details for PubMedID 31356864
Deletion of the glucagon receptor gene before and after experimental diabetes reveals differential protection from hyperglycemia
2018; 17: 28–38
Mice with congenital loss of the glucagon receptor gene (Gcgr-/- mice) remain normoglycemic in insulinopenic conditions, suggesting that unopposed glucagon action is the driving force for hyperglycemia in Type-1 Diabetes Mellitus (T1DM). However, chronic loss of GCGR results in a neomorphic phenotype that includes hormonal signals with hypoglycemic activity. We combined temporally-controlled GCGR deletion with pharmacological treatments to dissect the direct contribution of GCGR signaling to glucose control in a common mouse model of T1DM.We induced experimental T1DM by injecting the beta-cell cytotoxin streptozotocin (STZ) in mice with congenital or temporally-controlled Gcgr loss-of-function using tamoxifen (TMX).Disruption of Gcgr expression, using either an inducible approach in adult mice or animals with congenital knockout, abolished the response to a long-acting Gcgr agonist. Mice with either developmental Gcgr disruption or inducible deletion several weeks before STZ treatment maintained normoglycemia. However, mice with inducible knockout of the Gcgr one week after the onset of STZ diabetes had only partial correction of hyperglycemia, an effect that was reversed by GLP-1 receptor blockade. Mice with Gcgr deletion for either 2 or 6 weeks had similar patterns of gene expression, although the changes were generally larger with longer GCGR knockout.These findings demonstrate that the effects of glucagon to mitigate diabetic hyperglycemia are not through acute signaling but require compensations that take weeks to develop.
View details for DOI 10.1016/j.molmet.2018.07.012
View details for Web of Science ID 000447687500003
View details for PubMedID 30170980
View details for PubMedCentralID PMC6197675
A synbiotic composed of Lactobacillus fermentum CECT5716 and FOS prevents the development of fatty acid liver and glycemic alterations in rats fed a high fructose diet associated with changes in the microbiota
MOLECULAR NUTRITION & FOOD RESEARCH
2017; 61 (8)
We investigated the effect of a high fructose diet (HFD) on Sprague Dawley rats and the impact of a synbiotic composed of Lactobacillus fermentum CECT5716 and fructooligosaccharides. Feeding the HFD for 5 weeks resulted in liver steatosis and insulin resistance but not obesity. These changes were associated with increased production of short-chain fatty acids and increased Bacteroidetes in feces, with an augmented Bacteroidetes/Firmicutes ratio, among other changes in the microbiota. In addition, barrier function was weakened, with increased LPS plasma levels. These data are consistent with increased fructose availability in the distal gut due to saturation of absorptive mechanisms, leading to dysbiosis, endotoxemia, hepatic steatosis, and insulin resistance. Treatment with the synbiotic prevented some of the pathological effects, so that treated rats did not develop steatosis or systemic inflammation, while dysbiosis and barrier function were greatly ameliorated. In addition, the synbiotic had hypolipidemic effects. The synbiotic composed by L. fermentum CECT5716 and fructooligosaccharides has beneficial effects in a model of metabolic syndrome induced by a HFD, suggesting it might be clinically useful in this type of condition, particularly considering that high fructose intake has been related to metabolic syndrome in humans.
View details for DOI 10.1002/mnfr.201600622
View details for Web of Science ID 000407713400025
View details for PubMedID 28463404
The Circadian Clock Regulates Adipogenesis by a Per3 Crosstalk Pathway to Klf15.
2017; 21 (9): 2367–75
The generation of new adipocytes from precursor cells (adipogenesis) has implications for systemic metabolism and is a commonly used model for studying the process of cell differentiation in vitro. Previous studies from us and others suggested that the peripheral circadian clock can influence adipogenesis in vitro, but the mechanisms driving this activity and the relevance for adipogenesis in vivo are unknown. Here we reveal that mouse adipocyte precursor cells (APCs) contain a circadian clock that oscillates in vivo. We expose context-specific features of the clock in APCs: expression of the canonical core clock component Per1 does not significantly oscillate, whereas the lesser-understood paralog Per3 has a prominent rhythm. We discovered that deletion of Per3 promotes adipogenesis in vivo by a clock output pathway in which PER3 and BMAL1 directly regulate Klf15 expression. These findings demonstrate that Per3 has a major role in the APC clock and regulates adipogenesis in vivo.
View details for PubMedID 29186676
Human adipose tissue expresses intrinsic circadian rhythm in insulin sensitivity
2016; 30 (9): 3117-3123
In humans, insulin sensitivity varies according to time of day, with decreased values in the evening and at night. Mechanisms responsible for the diurnal variation in insulin sensitivity are unclear. We investigated whether human adipose tissue (AT) expresses intrinsic circadian rhythms in insulin sensitivity that could contribute to this phenomenon. Subcutaneous and visceral AT biopsies were obtained from extremely obese participants (body mass index, 41.8 ± 6.3 kg/m(2); 46 ± 11 y) during gastric-bypass surgery. To assess the rhythm in insulin signaling, AKT phosphorylation was determined every 4 h over 24 h in vitro in response to different insulin concentrations (0, 1, 10, and 100 nM). Data revealed that subcutaneous AT exhibited robust circadian rhythms in insulin signaling (P < 0.00001). Insulin sensitivity reached its maximum (acrophase) around noon, being 54% higher than during midnight (P = 0.009). The amplitude of the rhythm was positively correlated with in vivo sleep duration (r = 0.53; P = 0.023) and negatively correlated with in vivo bedtime (r = -0.54; P = 0.020). No circadian rhythms were detected in visceral AT (P = 0.643). Here, we demonstrate the relevance of the time of the day for how sensitive AT is to the effects of insulin. Subcutaneous AT shows an endogenous circadian rhythm in insulin sensitivity that could provide an underlying mechanism for the daily rhythm in systemic insulin sensitivity.-Carrasco-Benso, M. P., Rivero-Gutierrez, B., Lopez-Minguez, J., Anzola, A., Diez-Noguera, A., Madrid, J. A., Lujan, J. A., Martínez-Augustin, O., Scheer, F. A. J. L., Garaulet, M. Human adipose tissue expresses intrinsic circadian rhythm in insulin sensitivity.
View details for DOI 10.1096/fj.201600269RR
View details for Web of Science ID 000382816300012
View details for PubMedID 27256623
View details for PubMedCentralID PMC5001513
Diet-Induced Obese Mice Retain Endogenous Leptin Action
2015; 21 (6): 877-882
Obesity is characterized by hyperleptinemia and decreased response to exogenous leptin. This has been widely attributed to the development of leptin resistance, a state of impaired leptin signaling proposed to contribute to the development and persistence of obesity. To directly determine endogenous leptin activity in obesity, we treated lean and obese mice with a leptin receptor antagonist. The antagonist increased feeding and body weight (BW) in lean mice, but not in obese models of leptin, leptin receptor, or melanocortin-4 receptor deficiency. In contrast, the antagonist increased feeding and BW comparably in lean and diet-induced obese (DIO) mice, an increase associated with decreased hypothalamic expression of Socs3, a primary target of leptin. These findings demonstrate that hyperleptinemic DIO mice retain leptin suppression of feeding comparable to lean mice and counter the view that resistance to endogenous leptin contributes to the persistence of DIO in mice.
View details for DOI 10.1016/j.cmet.2015.04.015
View details for Web of Science ID 000355673700012
View details for PubMedID 25980347
View details for PubMedCentralID PMC4456263
Stain-free detection as loading control alternative to Ponceau and housekeeping protein immunodetection in Western blotting
2014; 467: 1-3
It is currently a routine practice to require a measurement of a housekeeping reference, including actin, glyceraldehyde-3-phosphate dehydrogenase, β-tubulin, among others, in Western blots, as it is the rule in RNA blots. Reversible Ponceau staining has been applied successfully to check equal loading of gels. Here we test a new technique, with the Stain-Free gels from Bio-Rad, against both Ponceau staining and housekeeping protein immunodetection under different conditions. Our results show that Stain-Free gels outperform Ponceau staining and that both are more consistent than housekeeping proteins as a loading control.
View details for DOI 10.1016/j.ab.2014.08.027
View details for Web of Science ID 000345059900001
View details for PubMedID 25193447
Food Derived Bioactive Peptides and Intestinal Barrier Function
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
2014; 15 (12): 22857-22873
A wide range of food-derived bioactive peptides have been shown to exert health-promoting actions and are therefore considered functional foods or nutraceuticals. Some of these actions are related to the maintenance, reinforcement or repairment of the intestinal barrier function (IBF) whose role is to selectively allow the absorption of water, nutrients and ions while preventing the influx of microorganisms from the intestinal lumen. Alterations in the IBF have been related to many disorders, such as inflammatory bowel disease or metabolic syndrome. Components of IBF are the intestinal epithelium, the mucus layer, secretory immunoglobulin A and cells of the innate and adaptive immune systems. Here we review the effects of food derived bioactive peptides on these IBF components. In vitro and in vivo effects, both in healthy and disease states, have been reviewed. Although limited, the available information indicates a potential for food-derived peptides to modify IBF and to contribute to disease treatment, but further research is needed to better isolate responsible peptides, and to help define their mode of action.
View details for DOI 10.3390/ijms151222857
View details for Web of Science ID 000346797400077
View details for PubMedID 25501338
View details for PubMedCentralID PMC4284742