Mcam stabilizes luminal progenitor breast cancer phenotypes via Ck2 control and Src/Akt/Stat3 attenuation.

Breast cancers are categorized into subtypes with distinctive therapeutic vulnerabilities and prognoses based on their expression of clinically targetable receptors and gene expression patterns mimicking different cell types of the normal gland. Here, we tested the role of Mcam in breast cancer cell state control and tumorigenicity in a luminal progenitor-like murine tumor cell line (Py230) that exhibits lineage and tumor subtype plasticity. Mcam knockdown Py230 cells show augmented Stat3 and Pi3K/Akt activation associated with a lineage state switch away from a hormone-sensing/luminal progenitor state toward alveolar and basal cell related phenotypes that were refractory to growth inhibition by the anti-estrogen therapeutic, tamoxifen. Inhibition of Stat3, or the upstream activator Ck2, reversed these cell state changes. Mcam binds Ck2 and acts as a regulator of Ck2 substrate utilization across multiple mammary tumor cell lines. In Py230 cells this activity manifests as increased mesenchymal morphology, migration, and Src/Fak/Mapk/Paxillin adhesion complex signaling in vitro, in contrast to Mcam's reported roles in promoting mesenchymal phenotypes. In vivo, Mcam knockdown reduced tumor growth and take rate and inhibited cell state transition to Sox10+/neural crest like cells previously been associated with tumor aggressiveness. This contrasts with human luminal breast cancers where MCAM copy number loss is highly coupled to Cyclin D amplification, increased proliferation, and the more aggressive Luminal B subtype. Together these data indicate a critical role for Mcam and its regulation of Ck2 in control of breast cancer cell state plasticity with implications for progression, evasion of targeted therapies and combination therapy design.

CRIPTO promotes extracellular vesicle uptake and activation of cancer associated fibroblasts.

Expression of CRIPTO, a factor involved in embryonic stem cells, fetal development, and wound healing, is tied to poor prognosis in multiple cancers. Prior studies in triple negative breast cancer (TNBC) models showed CRIPTO blockade inhibits tumor growth and dissemination. Here, we uncover a previously unidentified role for CRIPTO in orchestrating tumor-derived extracellular vesicle (TEV) uptake and fibroblast activation through discrete mechanisms. We found a novel mechanism by which CRIPTO drives aggressive TNBC phenotypes, involving CRIPTO-laden TEVs that program stromal fibroblasts, toward cancer associated fibroblast cell states, which in turn prompt tumor cell invasion. CRIPTO-bearing TEVs exhibited markedly elevated uptake in target fibroblasts and activated SMAD2/3 through NODAL-independent and - dependent mechanisms, respectively. Engineered expression of CRIPTO on EVs enhanced the delivery of bioactive molecules. In vivo , CRIPTO levels dictated TEV uptake in mouse lungs, a site of EV-regulated premetastatic niches important for breast cancer dissemination. These discoveries reveal a novel role for CRIPTO in coordinating heterotypic cellular crosstalk which offers novel insights into breast cancer progression, delivery of therapeutic molecules, and new, potentially targetable mechanisms of heterotypic cellular communication between tumor cells and the TME.

Growth Differentiation Factor 15 and Diet Quality Trajectory Interact to Determine Frailty Incidence among Middle-Aged Urban Adults.

BACKGROUND: Elevated plasma growth differentiation factor 15 (GDF15) and poor diet quality may be associated with increased frailty incidence, although their interactive associations have not been assessed in urban middle-aged adults. OBJECTIVES: We aimed to examine GDF15 and its interactive association with diet quality in relation to frailty incidence among a sample of middle-aged urban adults. METHODS: The relationship between GDF15 and diet quality trajectories in relation to incident frailty was examined in a longitudinal study of participants in the Healthy Aging in Neighborhoods of Diversity across the Life Span (2004-2017). Serum GDF15 concentration and frailty incidence were primary exposure and outcome, respectively. Group-based trajectory models were used to assess diet quality trajectories (≤3 visits/participant, N = 945, N' = 2247 observations) using the Healthy Eating Index 2010 version (HEI-2010), Dietary Inflammatory Index, and mean adequacy ratio (MAR). Cox proportional hazards models were used, testing interactive associations of GDF15 and diet quality trajectories with frail/prefrail incidence (N = 400 frailty-free at first visit, N' = 604 observations, n = 168 incident frail/prefrail). RESULTS: Both elevated GDF15 and lower diet quality trajectories were associated with a lower probability of remaining nonfrail (≤13 y follow-up). Among females, the "high diet quality" HEI-2010 trajectory had a hazard ratio (HR) of 0.15 [95% confidence interval (CI): 0.04, 0.54; P = 0.004; fully adjusted model] when compared with the "low diet quality" trajectory group. Among males only, there was an antagonistic interaction between lower HEI-2010 trajectory and elevated GDF15. Specifically, the HR for GDF15-frailty in the higher diet quality trajectory group (high/medium combined), and among males, was 2.69 (95% CI: 1.06, 6.62; P = 0.032), whereas among the lower diet quality trajectory group, the HR was 0.94 (95% CI: 0.49, 1.80; P = 0.86). Elevated GDF15 was independently associated with frailty among African American adults. CONCLUSIONS: Pending replication, we found an antagonistic interaction between GDF15 and HEI-2010 trajectory in relation to frailty incidence among males.

Safety, feasibility, and impact on the gut microbiome of kefir administration in critically ill adults.

BACKGROUND: Dysbiosis of the gut microbiome is frequent in the intensive care unit (ICU), potentially leading to a heightened risk of nosocomial infections. Enhancing the gut microbiome has been proposed as a strategic approach to mitigate potential adverse outcomes. While prior research on select probiotic supplements has not successfully shown to improve gut microbial diversity, fermented foods offer a promising alternative. In this open-label phase I safety and feasibility study, we examined the safety and feasibility of kefir as an initial step towards utilizing fermented foods to mitigate gut dysbiosis in critically ill patients. METHODS: We administered kefir in escalating doses (60 mL, followed by 120 mL after 12 h, then 240 mL daily) to 54 critically ill patients with an intact gastrointestinal tract. To evaluate kefir's safety, we monitored for gastrointestinal symptoms. Feasibility was determined by whether patients received a minimum of 75% of their assigned kefir doses. To assess changes in the gut microbiome composition following kefir administration, we collected two stool samples from 13 patients: one within 72 h of admission to the ICU and another at least 72 h after the first stool sample. RESULTS: After administering kefir, none of the 54 critically ill patients exhibited signs of kefir-related bacteremia. No side effects like bloating, vomiting, or aspiration were noted, except for diarrhea in two patients concurrently on laxatives. Out of the 393 kefir doses prescribed for all participants, 359 (91%) were successfully administered. We were able to collect an initial stool sample from 29 (54%) patients and a follow-up sample from 13 (24%) patients. Analysis of the 26 paired samples revealed no increase in gut microbial α-diversity between the two timepoints. However, there was a significant improvement in the Gut Microbiome Wellness Index (GMWI) by the second timepoint (P = 0.034, one-sided Wilcoxon signed-rank test); this finding supports our hypothesis that kefir administration can improve gut health in critically ill patients. Additionally, the known microbial species in kefir were found to exhibit varying levels of engraftment in patients' guts. CONCLUSIONS: Providing kefir to critically ill individuals is safe and feasible. Our findings warrant a larger evaluation of kefir's safety, tolerability, and impact on gut microbiome dysbiosis in patients admitted to the ICU. TRIAL REGISTRATION: NCT05416814; trial registered on June 13, 2022.

GDF15 and its association with cognitive performance over time in a longitudinal study of middle-aged urban adults.

Serum GDF15 levels are correlated with multiple neurodegenerative diseases. Few studies have tested this marker's association with middle-aged cognitive performance over time, and whether race affects this association is unknown. We examined associations of initial serum GDF15 concentrations with longitudinal cognitive performance, spanning domains of global mental status, visual and verbal memory, attention, fluency, and executive function in a sub-sample of adults participating in the Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) study (n = 776, Agev1:30-66y, 45.6 % male, 57.0 % African American, 43.0 % below poverty). This analysis consisted of mixed-effects regression models applied to the total selected sample, while also stratifying the analyses by race in the main analyses and further stratifying by sex, age group and poverty status in an exploratory analysis. Our main findings, which passed multiple testing and covariate-adjustment, indicated that GDF15 was associated with poorer baseline performance on several cognitive tests, including animal fluency [overall sample: (Model 1: γ0 ± SE: -0.664 ± 0.208, P < 0.001; Model 2, γ0 ± SE: -0.498 ± 0.217, P < 0.05)]. Among White adults, GDF15 was linked to poorer performance on a brief test of attention (Model 1: γ0 ± SE: -0.426 ± 0.126, P < 0.001; Model 2, γ0 ± SE: -0.281 ± 0.139, P < 0.05); and Trailmaking test, part B (Model 1: γ0 ± SE: +0.129 ± 0.040, P < 0.001; Model 2, γ0 ± SE: +0.089 ± 0.041, P < 0.05), the latter being also linked to higher GDF15 among individuals living below poverty. Among women, GDF15 was associated with poor global mental status (Normalized MMSE: Model 1: γ0 ± SE: -2.617 ± 0.746, P < 0.001; Model 2: γ0 ± SE: -1.729 ± 0.709, P < 0.05). GDF15 was not associated with decline on any of the 11 cognitive test scores considered in âˆ¼ 4 years of follow-up. In sum, we detected cross-sectional associations between GDF15 and cognition, although GDF15 did not predict rate of change in cognitive performance over time among a sample of middle-aged adults. More longitudinal studies are needed to address the clinical utility of this biomarker for early cognitive defects.

Whence CRIPTO: The Reemergence of an Oncofetal Factor in 'Wounds' That Fail to Heal.

There exists a set of factors termed oncofetal proteins that play key roles in ontogeny before they decline or disappear as the organism's tissues achieve homeostasis, only to then re-emerge in cancer. Although the unique therapeutic potential presented by such factors has been recognized for more than a century, their clinical utility has yet to be fully realized1. This review highlights the small signaling protein CRIPTO encoded by the tumor derived growth factor 1 (TDGF1/Tdgf1) gene, an oft cited oncofetal protein whose presence in the cancer literature as a tumor promoter, diagnostic marker and viable therapeutic target continues to grow. We touch lightly on features well established and well-reviewed since its discovery more than 30 years ago, including CRIPTO's early developmental roles and modulation of SMAD2/3 activation by a selected set of transforming growth factor ß (TGF-ß) family ligands. We predominantly focus instead on more recent and less well understood additions to the CRIPTO signaling repertoire, on its potential upstream regulators and on new conceptual ground for understanding its mode of action in the multicellular and often stressful contexts of neoplastic transformation and progression. We ask whence it re-emerges in cancer and where it 'hides' between the time of its fetal activity and its oncogenic reemergence. In this regard, we examine CRIPTO's restriction to rare cells in the adult, its potential for paracrine crosstalk, and its emerging role in inflammation and tissue regeneration-roles it may reprise in tumorigenesis, acting on subsets of tumor cells to foster cancer initiation and progression. We also consider critical gaps in knowledge and resources that stand between the recent, exciting momentum in the CRIPTO field and highly actionable CRIPTO manipulation for cancer therapy and beyond.

CRIPTO antagonist ALK4L75A-Fc inhibits breast cancer cell plasticity and adaptation to stress.

BACKGROUND: CRIPTO is a multi-functional signaling protein that promotes stemness and oncogenesis. We previously developed a CRIPTO antagonist, ALK4L75A-Fc, and showed that it causes loss of the stem cell phenotype in normal mammary epithelia suggesting it may similarly inhibit CRIPTO-dependent plasticity in breast cancer cells. METHODS: We focused on two triple negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) to measure the effects of ALK4L75A-Fc on cancer cell behavior under nutrient deprivation and endoplasmic reticulum stress. We characterized the proliferation and migration of these cells in vitro using time-lapse microscopy and characterized stress-dependent changes in the levels and distribution of CRIPTO signaling mediators and cancer stem cell markers. We also assessed the effects of ALK4L75A-Fc on proliferation, EMT, and stem cell markers in vivo as well as on tumor growth and metastasis using inducible lentiviral delivery or systemic administration of purified ALK4L75A-Fc, which represents a candidate therapeutic approach. RESULTS: ALK4L75A-Fc inhibited adaptive responses of breast cancer cells under conditions of nutrient and ER stress and reduced their proliferation, migration, clonogenicity, and expression of EMT and cancer stem cell markers. ALK4L75A-Fc also inhibited proliferation of human breast cancer cells in stressed tumor microenvironments in xenografts and reduced both primary tumor size and metastatic burden. CONCLUSIONS: Cancer cell adaptation to stresses such as nutrient deprivation, hypoxia, and chemotherapy can critically contribute to dormancy, metastasis, therapy resistance, and recurrence. Identifying mechanisms that govern cellular adaptation, plasticity, and the emergence of stem-like cancer cells may be key to effective anticancer therapies. Results presented here indicate that targeting CRIPTO with ALK4L75A-Fc may have potential as such a therapy since it inhibits breast cancer cell adaptation to microenvironmental challenges and associated stem-like and EMT phenotypes.

Association between GDF15, poverty and mortality in urban middle-aged African American and white adults.

Mortality disparities are influenced by race and poverty. There is limited information about whether poverty influences biologic markers of mortality risk. Emerging data suggests that growth differentiation factor 15 (GDF15) is associated with mortality; however, the interplay between GDF15, sociodemographic factors and mortality is not known. We sought to evaluate the interactions between GDF15 and sex, race and poverty status on mortality. Serum GDF15 was measured in 1036 African American and white middle-aged men and women above and below 125% of the Federal poverty status from the Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) study. Multivariable adjusted Cox regression models were used to assess the association between log-transformed GDF15 (logGDF15) and 12-year mortality outcomes (all-cause, cardiovascular- and cancer-specific outcomes) and interactions with sex, race and poverty status. Likelihood ratio tests were used to assess significance of the interaction terms. Median GDF15 was 655.2 pg/mL (IQR = 575.1). During 12.2 years of follow-up, 331 died of which 94 cardiovascular- and 87 were cancer-specific deaths. One unit of increase in logGDF15 was associated with a hazard ratio for all-cause mortality, cardiovascular- and cancer-specific mortality of 2.26 (95% confidence interval [CI], 1.94-2.64), 2.74 (95%CI, 2.06-3.63) and 1.41 (95%CI, 1.00-2.00), respectively. There was an interaction between logGDF15 and poverty status on all-cause mortality (p<0.05). The GDF15×poverty status interaction term improved model calibration for all-cause mortality. Our study provides the first evidence that the effect of elevated GDF15 on all-cause mortality is modified by poverty status.

Extracellular vesicles in diabetes mellitus induce alterations in endothelial cell morphology and migration.

BACKGROUND: Inflammation-related atherosclerotic peripheral vascular disease is a major end organ complication of diabetes mellitus that results in devastating morbidity and mortality. Extracellular vesicles (EVs) are nano-sized particles that contain molecular cargo and circulate in the blood. Here, we examined EV protein cargo from diabetic individuals and whether these EVs cause functional changes in endothelial cells. METHODS: We quantified inflammatory protein levels in plasma-derived EVs from a longitudinal cohort of euglycemic and diabetic individuals and used in vitro endothelial cell biological assays to assess the functional effects of these EVs with samples from a cross-sectional cohort. RESULTS: We found several significant associations between EV inflammatory protein levels and diabetes status. The angiogenic factor, vascular endothelial growth factor A (VEGF-A), was associated with diabetes status in our longitudinal cohort. Those with diabetes mellitus had higher EV VEGF-A levels compared to euglycemic individuals. Additionally, EV levels of VEGF-A were significantly associated with homeostatic model assessment of insulin resistance (HOMA-IR) and ß-cell function (HOMA-B). To test whether EVs with different inflammatory cargo can demonstrate different effects on endothelial cells, we performed cell migration and immunofluorescence assays. We observed that EVs from diabetic individuals increased cell lamellipodia formation and migration when compared to EVs from euglycemic individuals. CONCLUSIONS: Higher levels of inflammatory proteins were found in EVs from diabetic individuals. Our data implicate EVs as playing important roles in peripheral vascular disease that occur in individuals with diabetes mellitus and suggest that EVs may serve as an informative diagnostic tool for the disease.

Biological membranes in EV biogenesis, stability, uptake, and cargo transfer: an ISEV position paper arising from the ISEV membranes and EVs workshop.

Paracrine and endocrine roles have increasingly been ascribed to extracellular vesicles (EVs) generated by multicellular organisms. Central to the biogenesis, content, and function of EVs are their delimiting lipid bilayer membranes. To evaluate research progress on membranes and EVs, the International Society for Extracellular Vesicles (ISEV) conducted a workshop in March 2018 in Baltimore, Maryland, USA, bringing together key opinion leaders and hands-on researchers who were selected on the basis of submitted applications. The workshop was accompanied by two scientific surveys and covered four broad topics: EV biogenesis and release; EV uptake and fusion; technologies and strategies used to study EV membranes; and EV transfer and functional assays. In this ISEV position paper, we synthesize the results of the workshop and the related surveys to outline important outstanding questions about EV membranes and describe areas of consensus. The workshop discussions and survey responses reveal that while much progress has been made in the field, there are still several concepts that divide opinion. Good consensus exists in some areas, including particular aspects of EV biogenesis, uptake and downstream signalling. Areas with little to no consensus include EV storage and stability, as well as whether and how EVs fuse with target cells. Further research is needed in these key areas, as a better understanding of membrane biology will contribute substantially towards advancing the field of extracellular vesicles.

Association of Extracellular Vesicle Protein Cargo with Race and Clinical Markers of Mortality.

Differential mortality rates remain a significant health disparity in the United States, suggesting the need to investigate novel potential molecular markers associated with mortality. Extracellular vesicles (EVs), including exosomes, microvesicles and apoptotic bodies, are lipid-bound vesicles secreted by cells into the circulation. EVs mediate intercellular communication by shuttling functional signaling molecules as cargo. EV characteristics by race in the context of mortality risk factors have not been described. We isolated plasma EVs from a cross-sectional cohort of African Americans (AA) and whites and found no significant differences in EV size, distribution or concentration between race or by sex. However, EV cargo showed increased levels of phospho-p53, total p53, cleaved caspase 3, ERK1/2 and phospho-AKT in white individuals compared to AAs. phospho-IGF-1R levels were significantly higher in females compared to males. EV concentration was significantly associated with several clinical mortality risk factors: high-sensitivity C-reactive protein (hsCRP), homeostatic model assessment of insulin resistance (HOMA-IR), alkaline phosphatase, body mass index, waist circumference and pulse pressure. The association of EV proteins with mortality markers were dependent on race. These data suggest that EV cargo can differ by race and sex and is associated with mortality risk factors.

Comparing 3D ultrastructure of presynaptic and postsynaptic mitochondria.

Serial-section electron microscopy such as FIB-SEM (focused ion beam scanning electron microscopy) has become an important tool for neuroscientists to trace the trajectories and global architecture of neural circuits in the brain, as well as to visualize the 3D ultrastructure of cellular organelles in neurons. In this study, we examined 3D features of mitochondria in electron microscope images generated from serial sections of four regions of mouse brains: nucleus accumbens (NA), hippocampal CA1, somatosensory cortex and dorsal cochlear nucleus (DCN). We compared mitochondria in the presynaptic terminals to those in the postsynaptic/dendritic compartments, and we focused on the shape and size of mitochondria. A common feature of mitochondria among the four brain regions is that presynaptic mitochondria generally are small and short, and most of them do not extend beyond presynaptic terminals. In contrast, the majority of postsynaptic/dendritic mitochondria are large and many of them spread through significant portions of the dendrites. Comparing among the brain areas, the cerebral cortex and DCN have even larger postsynaptic/dendritic mitochondria than the NA and CA1. Our analysis reveals that mitochondria in neurons are differentially sized and arranged according to their subcellular locations, suggesting a spatial organizing principle of mitochondria at the synapse.

Altered Extracellular Vesicle Concentration, Cargo, and Function in Diabetes.

Type 2 diabetes is a chronic age-associated degenerative metabolic disease that reflects relative insulin deficiency and resistance. Extracellular vesicles (EVs) (exosomes, microvesicles, and apoptotic bodies) are small (30-400 nm) lipid-bound vesicles capable of shuttling functional proteins, nucleic acids, and lipids as part of intercellular communication systems. Recent studies in mouse models and in cell culture suggest that EVs may modulate insulin signaling. Here, we designed cross-sectional and longitudinal cohorts of euglycemic participants and participants with prediabetes or diabetes. Individuals with diabetes had significantly higher levels of EVs in their circulation than euglycemic control participants. Using a cell-specific EV assay, we identified that levels of erythrocyte-derived EVs are higher with diabetes. We found that insulin resistance increases EV secretion. Furthermore, the levels of insulin signaling proteins were altered in EVs from individuals with high levels of insulin resistance and ß-cell dysfunction. Moreover, EVs from individuals with diabetes were preferentially internalized by circulating leukocytes. Cytokine levels in the media and in EVs were higher from monocytes incubated with diabetic EVs. Microarray of these leukocytes revealed altered gene expression pathways related to cell survival, oxidative stress, and immune function. Collectively, these results suggest that insulin resistance increases the secretion of EVs, which are preferentially internalized by leukocytes, and alters leukocyte function.

Age-Related Changes in Plasma Extracellular Vesicle Characteristics and Internalization by Leukocytes.

Cells release lipid-bound extracellular vesicles (EVs; exosomes, microvesicles and apoptotic bodies) containing proteins, lipids and RNAs into the circulation. Vesicles mediate intercellular communication between both neighboring and distant cells. There is substantial interest in using EVs as biomarkers for age-related diseases including cancer, and neurodegenerative, metabolic and cardiovascular diseases. The majority of research focuses on identifying differences in EVs when comparing disease states and matched controls. Here, we analyzed circulating plasma EVs in a cross-sectional and longitudinal study in order to address age-related changes in community-dwelling individuals. We found that EV concentration decreases with advancing age. Furthermore, EVs from older individuals were more readily internalized by B cells and increased MHC-II expression on monocytes compared with EVs from younger individuals, indicating that the decreased concentration of EVs with age may be due in part to increased internalization. EVs activated both monocytes and B cells, and activation of B cells by LPS enhanced EV internalization. We also report a relative stability of EV concentration and protein amount in individual subjects over time. Our data provide important information towards establishing a profile of EVs with human age, which will further aid in the development of EV-based diagnostics for aging and age-related diseases.

Mitochondria in hippocampal presynaptic and postsynaptic compartments differ in size as well as intensity.

Experimental observations have hinted that, in different compartments of a neuron, mitochondria can be different in their structure, behavior and activity. However, mitochondria have never been systematically compared at the subcellular level in neurons. Using electron microscopy, we analyzed several thousands of mitochondria in the synapses of rat hippocampal neurons in vitro and in vivo. We focused on examining the intensity and size of mitochondria as these structural features have been correlated to the activity of mitochondria. We compared mitochondria in the presynaptic compartment to those in the postsynaptic compartment. We found that, at least in the synapses of hippocampal neurons, presynaptic mitochondria are smaller in diameter and overall higher in intensity (darker) than postsynaptic mitochondria. Our finding highlights the need for developing technologies that would measure the activity of individual mitochondria at single-mitochondria resolution in real time.