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Background and ObjectivesWhile most maintenance dialysis patients exhibit initial seroresponse to vaccination, concerns remain regarding the durability of this antibody response. This study evaluated immunity over time.Design, setting, participants, and measurementsThis retrospective cohort study included maintenance dialysis patients from a midsize national dialysis provider who received a complete SARS-CoV-2 vaccine series and had at least one antibody titer checked after full vaccination. Immunoglobulin G spike antibodies (SAb-IgG) titers were assessed monthly with routine labs beginning after full vaccination and followed over time; the semiquantitative SAb-IgG titer reported a range between 0 and ≥ 20 U/L. Descriptive analyses compared trends over time by prior history of COVID-19 and type of vaccine received. Time-to-event analyses were conducted for the outcome of loss of seroresponse (SAb-IgG < 1 U/L or development of COVID-19). Cox proportional hazards regression was used to adjust for additional clinical characteristics of interest.ResultsAmong 1898 maintenance dialysis patients, 1567 (84%) had no prior history of COVID-19. Patients without a history of COVID-19 had declining titers over time. Among 441 BNT162b2/Pfizer recipients, median [IQR] SAb-IgG titer declined from 20 [5.99-20] U/L in month 1 to 1.30 [0.15-3.59] U/L by month 6. Among 779 mRNA-1273/Moderna recipients, median [IQR] SAb-IgG titer declined from 20 [20-20] in month 1 to 6.20 [1.74-20] by month 6. The 347 Ad26.COV2.S/Janssen recipients had a lower titer response than mRNA vaccine recipients over all time periods. In time-to-event analyses, Ad26.COV2.S/Janssen and mRNA-1273/Moderna recipients had the shortest and longest time to loss of seroresponse, respectively. The maximum titer reached in the first two months after full vaccination was predictive of the durability of the SAb-IgG seroresponse; patients with SAb-IgG titer 1-19.99 U/L were more likely to have loss of seroresponse compared to patients with SAb-IgG titer ≥ 20 U/L (HR 23.9 [95% CI: 16.1-35.5]).ConclusionsVaccine-induced seroresponse wanes over time among maintenance dialysis patients across vaccine types. Early titers after full vaccination predict the durability of seroresponse.

Extracellular vesicles (EVs) are secreted by many cell types and are increasingly investigated for their role in human diseases including cancer. Here we focus on the secretion and potential physiological function of non-pathological EVs secreted by polarized normal mammary epithelial cells. Using a transwell system to allow formation of epithelial polarity and EV collection from the apical versus basolateral compartments, we found that impaired secretion of EVs by knockdown of RAB27A or RAB27B suppressed the establishment of mammary epithelial polarity, and that addition of apical but not basolateral EVs suppressed epithelial polarity in a dose-dependent manner. This suggests that apical EV secretion contributes to epithelial polarity, and a possible mechanism is through removal of certain intracellular molecules. In contrast, basolateral but not apical EVs promoted migration of mammary epithelial cells in a motility assay. The protein contents of apical and basolateral EVs from MCF10A and primary human mammary epithelial cells were determined by mass spectrometry proteomic analysis, identifying apical-EV-enriched and basolateral-EV-enriched proteins that may contribute to different physiological functions. Most of these proteins differentially secreted by normal mammary epithelial cells through polarized EV release no longer showed polarized secretion in MCF10A-derived transformed epithelial cells. Our results suggest an essential role of EV secretion in normal mammary epithelial polarization and distinct protein contents and functions in apical versus basolateral EVs secreted by polarized mammary epithelia.

Reprogrammed glucose metabolism as a result of increased glycolysis and glucose uptake is a hallmark of cancer. Here we show that cancer cells can suppress glucose uptake by non-tumour cells in the pre-metastatic niche, by secreting vesicles that carry high levels of the miR-122 microRNA. High miR-122 levels in the circulation have been associated with metastasis in breast cancer patients and we show that cancer-cell-secreted miR-122 facilitates metastasis by increasing nutrient availability in the pre-metastatic niche. Mechanistically cancer-cell-derived miR-122 suppresses glucose uptake by niche cells in vitro and in vivo by downregulating the glycolytic enzyme pyruvate kinase (PKM). In vivo inhibition of miR-122 restores glucose uptake in distant organs, including brain and lungs, and decreases the incidence of metastasis. These results demonstrate that by modifying glucose utilization by recipient pre-metastatic niche cells, cancer-derived extracellular miR-122 is able to reprogram systemic energy metabolism to facilitate disease progression.

A growing body of work has shown that cancer metastasis is not a random spontaneous event; rather, it is the culmination of a cascade of priming steps through which a subpopulation of the tumor cells acquires invasive traits while readying a permissive environment, termed the "premetastatic niche," in which distant metastases can occur. Signals from the primary tumor mobilize and adapt immune cells as well as directly communicating with distant niche cells to induce a broad spectrum of adaptations in target organs, including the induction of angiogenesis, inflammation, extracellular matrix remodeling, and metabolic reprogramming. Together, these interactions facilitate the formation of a premetastatic niche composed of a variable mix of resident and recruited immune cells, endothelial cells, and stromal cells connected through a complex signaling network that we are only beginning to understand. Here, we summarize the latest findings on how cancer induces and guides the formation of this premetastatic niche as well as potential prognostic markers and therapeutic targets that may lead to a better understanding and effective treatment of metastatic disease. Clin Cancer Res; 22(15); 3725–33. ©2016 AACR.

MicroRNAs (miRNAs) are critical regulators of gene expression, and exert extensive impacts on development, physiology, and disease of eukaryotes. A high degree of parallelism is found in the molecular basis of miRNA biogenesis and action in plants and animals. Recent studies interestingly suggest a potential cross-kingdom action of plant-derived miRNAs, through dietary intake, in regulating mammalian gene expression. Although the source and scope of plant miRNAs detected in mammalian specimens remain controversial, these initial studies inspired us to determine whether plant miRNAs can be detected in Western human sera and whether these plant miRNAs are able to influence gene expression and cellular processes related to human diseases such as cancer. Here we found that Western donor sera contained the plant miRNA miR159, whose abundance in the serum was inversely correlated with breast cancer incidence and progression in patients. In human sera, miR159 was predominantly detected in the extracellular vesicles, and was resistant to sodium periodate oxidation suggesting the plant-originated 2'-O-methylation on the 3' terminal ribose. In breast cancer cells but not non-cancerous mammary epithelial cells, a synthetic mimic of miR159 was capable of inhibiting proliferation by targeting TCF7 that encodes a Wnt signaling transcription factor, leading to a decrease in MYC protein levels. Oral administration of miR159 mimic significantly suppressed the growth of xenograft breast tumors in mice. These results demonstrate for the first time that a plant miRNA can inhibit cancer growth in mammals.

Transforming growth factor beta (TGFβ) proteins are multitasking cytokines, in which high levels at tumor sites generally correlate with poor prognosis in human patients with cancer. Previously, it was reported that TGFβ downregulates the expression of ataxia telangiectasia–mutated (ATM) and mutS homolog 2 (MSH2) in breast cancer cells through an miRNA-mediated mechanism. In this study, expression of a panel of DNA-repair genes was examined, identifying breast cancer 1, early onset (BRCA1) as a target downregulated by TGFβ through the miR181 family. Correlations between the expression levels of TGFβ1 and the miR181/BRCA1 axis were observed in primary breast tumor specimens. By downregulating BRCA1, ATM, and MSH2, TGFβ orchestrates DNA damage response in certain breast cancer cells to induce a “BRCAness” phenotype, including impaired DNA-repair efficiency and synthetic lethality to the inhibition of poly (ADP-ribose) polymerase (PARP). Xenograft tumors with active TGFβ signaling exhibited resistance to the DNA-damaging agent doxorubicin but increased sensitivity to the PARP inhibitor ABT-888. Combination of doxorubicin with ABT-888 significantly improved the treatment efficacy in TGFβ-active tumors. Thus, TGFβ can induce “BRCAness” in certain breast cancers carrying wild-type BRCA genes and enhance the responsiveness to PARP inhibition, and the molecular mechanism behind this is characterized. Implications: These findings enable better selection of patients with sporadic breast cancer for PARP interventions, which have exhibited beneficial effects in patients carrying BRCA mutations. Mol Cancer Res; 12(11); 1597–609. ©2014 AACR.

Growing evidence links tumor progression with chronic inflammatory processes and dysregulated activity of various immune cells. In this study, we demonstrate that various types of macrophages internalize microvesicles, called exosomes, secreted by breast cancer and non-cancerous cell lines. Although both types of exosomes targeted macrophages, only cancer-derived exosomes stimulated NF-κB activation in macrophages resulting in secretion of pro-inflammatory cytokines such as IL-6, TNFα, GCSF, and CCL2. In vivo mouse experiments confirmed that intravenously injected exosomes are efficiently internalized by macrophages in the lung and brain, which correlated with upregulation of inflammatory cytokines. In mice bearing xenografted human breast cancers, tumor-derived exosomes were internalized by macrophages in axillary lymph nodes thereby triggering expression of IL-6. Genetic ablation of Toll-like receptor 2 (TLR2) or MyD88, a critical signaling adaptor in the NF-κB pathway, completely abolished the effect of tumor-derived exosomes. In contrast, inhibition of TLR4 or endosomal TLRs (TLR3/7/8/9) failed to abrogate NF-κB activation by exosomes. We further found that palmitoylated proteins present on the surface of tumor-secreted exosomes contributed to NF-κB activation. Thus, our results highlight a novel mechanism used by breast cancer cells to induce pro-inflammatory activity of distant macrophages through circulating exosomal vesicles secreted during cancer progression.

There is increasing evidence for the cancer stem cell model in which a subset of cancer cells possessing stem cell properties, referred to as tumor-initiating or cancer stem-like cells (CSCs), play crucial roles in multiple aspects of cancer. Recent studies have started to characterize the crucial role of various cytokines in the tumor microenvironment in regulating the fate of CSCs. In this review, we summarized some of the latest findings on cytokines that drive breast cancer stemness and their mechanisms of action. These cytokines, including IL-6, IL-8, CCL2 and TGF-β, are frequently elevated in breast tumors and may hold promise as potential therapeutic targets to eradicate CSCs. In combination with conventional chemotherapy and radiotherapy targeting rapidly proliferating cancer cells, intervention of the cancer stemness-driving cytokines may achieve additional benefits for breast cancer patients by suppressing CSC-promoted cancer progression, recurrence, and drug refractoriness.

SummaryCancer-secreted microRNAs (miRNAs) are emerging mediators of cancer-host crosstalk. Here we show that miR-105, which is characteristically expressed and secreted by metastatic breast cancer cells, is a potent regulator of migration through targeting the tight junction protein ZO-1. In endothelial monolayers, exosome-mediated transfer of cancer-secreted miR-105 efficiently destroys tight junctions and the integrity of these natural barriers against metastasis. Overexpression of miR-105 in nonmetastatic cancer cells induces metastasis and vascular permeability in distant organs, whereas inhibition of miR-105 in highly metastatic tumors alleviates these effects. miR-105 can be detected in the circulation at the premetastatic stage, and its levels in the blood and tumor are associated with ZO-1 expression and metastatic progression in early-stage breast cancer.

Human embryonic stem cell (hESc) lines are likely the in vitro equivalent of the pluripotent epiblast. hESc express high levels of the extracellular matrix (ECM) laminin integrin receptor α6β1 and consequently can adhere robustly and be propagated in an undifferentiated state on tissue culture plastic coated with the laminin rich basement membrane preparation, Matrigel, even in the absence of supporting fibroblasts. Such cultures represent a critical step in the development of more defined feeder free cultures of hESc; a goal deemed necessary for regenerative medical applications and have been used as the starting point in some differentiation protocols. However, on standard non-deformable tissue culture plastic hESc either fail or inadequately develop the structural/morphological organization of the epiblast in vivo. By contrast, growth of hESc on appropriately defined mechanically deformable polyacrylamide substrates permits recapitulation of many of these in vivo features. These likely herald differences in the precise nature of the integration of signal transduction pathways from soluble morphogens and represent an unexplored variable in hESc (fate) state space. In this chapter we describe how to establish viable hESc colonies on these functionalized polyacrylamide gels. We suggest this strategy as a prospective in vitro model of the genetics, biochemistry, and cell biology of pre- and early-gastrulation stage human embryos and the permissive and instructive roles that cellular and substrate mechanics might play in early embryonic cell fate decisions. Such knowledge should inform regenerative medical applications aimed at enabling or improving the differentiation of specific cell types from embryonic or induced embryonic stem cells.