Intraductal Transplantation Models of Human Pancreatic Ductal Adenocarcinoma Reveal Progressive Transition of Molecular Subtypes.

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal common malignancy, with little improvement in patient outcomes over the past decades. Recently, subtypes of pancreatic cancer with different prognoses have been elaborated; however, the inability to model these subtypes has precluded mechanistic investigation of their origins. Here, we present a xenotransplantation model of PDAC in which neoplasms originate from patient-derived organoids injected directly into murine pancreatic ducts. Our model enables distinction of the two main PDAC subtypes: intraepithelial neoplasms from this model progress in an indolent or invasive manner representing the classical or basal-like subtypes of PDAC, respectively. Parameters that influence PDAC subtype specification in this intraductal model include cell plasticity and hyperactivation of the RAS pathway. Finally, through intratumoral dissection and the direct manipulation of RAS gene dosage, we identify a suite of RAS-regulated secreted and membrane-bound proteins that may represent potential candidates for therapeutic intervention in patients with PDAC. SIGNIFICANCE: Accurate modeling of the molecular subtypes of pancreatic cancer is crucial to facilitate the generation of effective therapies. We report the development of an intraductal organoid transplantation model of pancreatic cancer that models the progressive switching of subtypes, and identify stochastic and RAS-driven mechanisms that determine subtype specification.See related commentary by Pickering and Morton, p. 1448.This article is highlighted in the In This Issue feature, p. 1426.

SOAT1 promotes mevalonate pathway dependency in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and new therapies are needed. Altered metabolism is a cancer vulnerability, and several metabolic pathways have been shown to promote PDAC. However, the changes in cholesterol metabolism and their role during PDAC progression remain largely unknown. Here we used organoid and mouse models to determine the drivers of altered cholesterol metabolism in PDAC and the consequences of its disruption on tumor progression. We identified sterol O-acyltransferase 1 (SOAT1) as a key player in sustaining the mevalonate pathway by converting cholesterol to inert cholesterol esters, thereby preventing the negative feedback elicited by unesterified cholesterol. Genetic targeting of Soat1 impairs cell proliferation in vitro and tumor progression in vivo and reveals a mevalonate pathway dependency in p53 mutant PDAC cells that have undergone p53 loss of heterozygosity (LOH). In contrast, pancreatic organoids lacking p53 mutation and p53 LOH are insensitive to SOAT1 loss, indicating a potential therapeutic window for inhibiting SOAT1 in PDAC.

Identification of Resistance Pathways Specific to Malignancy Using Organoid Models of Pancreatic Cancer.

PURPOSE: KRAS is mutated in the majority of pancreatic ductal adenocarcinoma. MAPK and PI3K-AKT are primary KRAS effector pathways, but combined MAPK and PI3K inhibition has not been demonstrated to be clinically effective to date. We explore the resistance mechanisms uniquely employed by malignant cells. EXPERIMENTAL DESIGN: We evaluated the expression and activation of receptor tyrosine kinases in response to combined MEK and AKT inhibition in KPC mice and pancreatic ductal organoids. In addition, we sought to determine the therapeutic efficacy of targeting resistance pathways induced by MEK and AKT inhibition in order to identify malignant-specific vulnerabilities. RESULTS: Combined MEK and AKT inhibition modestly extended the survival of KPC mice and increased Egfr and ErbB2 phosphorylation levels. Tumor organoids, but not their normal counterparts, exhibited elevated phosphorylation of ERBB2 and ERBB3 after MEK and AKT blockade. A pan-ERBB inhibitor synergized with MEK and AKT blockade in human PDA organoids, whereas this was not observed for the EGFR inhibitor erlotinib. Combined MEK and ERBB inhibitor treatment of human organoid orthotopic xenografts was sufficient to cause tumor regression in short-term intervention studies. CONCLUSIONS: Analyses of normal and tumor pancreatic organoids revealed the importance of ERBB activation during MEK and AKT blockade primarily in the malignant cultures. The lack of ERBB hyperactivation in normal organoids suggests a larger therapeutic index. In our models, pan-ERBB inhibition was synergistic with dual inhibition of MEK and AKT, and the combination of a pan-ERBB inhibitor with MEK antagonists showed the highest activity both in vitro and in vivo.

Organoid models for translational pancreatic cancer research.

Despite recent advances in the treatment of cancer, pancreatic ductal adenocarcinoma (PDAC) still retains the worst survival rate of common malignancies. Late diagnosis and lack of curative therapeutic options are the most pressing clinical problems for this disease. Therefore, there is a need for patient models and biomarkers that can be applied in the clinic to identify the most effective therapy for a patient. Pancreatic ductal organoids are ex-vivo models of PDAC that can be established from very small biopsies, enabling the study of localized, advanced, and metastatic patients. Organoids models have been applied to pancreatic cancer research and offer a promising platform for precision medicine approaches.

Generation and Culture of Tumor and Metastatic Organoids from Murine Models of Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy that is refractory to all current therapies. Research into the mechanisms driving this cancer is the key to developing better diagnostic and treatment options which are urgently needed in the clinic. Genetically engineered mouse models of PDA have been valuable research tools, enabling studies of all stages of PDA progression. However, these models are difficult and time-consuming to breed, and engineering further mutations into these models requires additional time. Recently, organoid cultures of PDA have emerged as alternative models for this disease. Organoids can be rapidly generated from mouse models of PDA and enable genetic and biochemical perturbation of all stages of PDA progression. Here, we describe the generation and propagation of organoid models from PDA tumors and metastases harvested from genetically engineered mouse models.

Generation and Culture of Human Pancreatic Ductal Adenocarcinoma Organoids from Resected Tumor Specimens.

The recent development of human organoids as patient-specific models of pancreatic ductal adenocarcinoma (PDA) has helped set the stage for a new era of personalized medicine. Organoids can be generated from a resected PDA tumor in as little as 2-4 weeks, and are amenable to therapeutic screening as well as genetic and biochemical perturbation. Moreover, because these models promote the propagation of the neoplastic PDA cells at the expense of the stromal cells, transcriptome and genome-wide sequencing of organoids offers an unprecedented view of the genetic and expression changes occurring in the neoplastic cells of individual tumors. Here, we describe methods to generate PDA organoid cultures from resected human tumor specimens. We also describe how to propagate, cryopreserve, and thaw human PDA organoid cultures.

Testing Susceptibility of Patient-Derived Organoid Cultures to Therapies: Pharmacotyping.

Increasingly, patient models of disease are being utilized to facilitate precision medicine approaches through molecular characterization or direct chemotherapeutic testing. Organoids, 3-dimensional (3D) cultures of neoplastic cells derived from primary tumor specimens, represent an ideal platform for these types of studies because benchtop protocols previously developed for 2-dimensional cell lines can be adapted for use. These protocols include directly testing the survival of these organoid cultures when exposed to clinically relevant chemotherapeutic agents, a process we have called pharmacotyping. In this protocol, established tumor-derived organoid cultures are dissociated into single cells, plated in a 3D gel matrix, and exposed to pharmacologic agents. While our protocol has been developed for use with patient-derived pancreatic ductal adenocarcinoma organoids, with minor modifications to the dissociation and medium conditions, this protocol could be adapted for use with a wide range of organoid cultures. We further describe our standard ATP-based assay to determine cellular survival. This protocol can be scaled for use in high-throughput assays.

Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer.

Pancreatic stellate cells (PSCs) differentiate into cancer-associated fibroblasts (CAFs) that produce desmoplastic stroma, thereby modulating disease progression and therapeutic response in pancreatic ductal adenocarcinoma (PDA). However, it is unknown whether CAFs uniformly carry out these tasks or if subtypes of CAFs with distinct phenotypes in PDA exist. We identified a CAF subpopulation with elevated expression of α-smooth muscle actin (αSMA) located immediately adjacent to neoplastic cells in mouse and human PDA tissue. We recapitulated this finding in co-cultures of murine PSCs and PDA organoids, and demonstrated that organoid-activated CAFs produced desmoplastic stroma. The co-cultures showed cooperative interactions and revealed another distinct subpopulation of CAFs, located more distantly from neoplastic cells, which lacked elevated αSMA expression and instead secreted IL6 and additional inflammatory mediators. These findings were corroborated in mouse and human PDA tissue, providing direct evidence for CAF heterogeneity in PDA tumor biology with implications for disease etiology and therapeutic development.

Model organoids provide new research opportunities for ductal pancreatic cancer.

We recently established organoid models from normal and neoplastic murine and human pancreas tissues. These organoids exhibit ductal- and disease stage-specific characteristics and, after orthotopic transplantation, recapitulate the full spectrum of tumor progression. Pancreatic organoid technology provides a novel platform for the study of tumor biology and the discovery of potential biomarkers, therapeutics, and personalized medicine strategies.

Modeling pancreatic cancer with organoids.

Pancreatic ductal adenocarcinoma (PDA) is a highly lethal malignancy for which new treatment and diagnostic approaches are urgently needed. In order for such breakthroughs to be discovered, researchers require systems that accurately model the development and biology of PDA. While cell lines, genetically engineered murine models, and xenografts have all led to valuable clinical insights, organotypic culture models have emerged as tractable systems to recapitulate the complex three-dimensional organization of PDA. Recently, multiple methods for modeling PDA using organoids have been reported. This review aims to summarize these organoid methods in the context of other PDA models. While each model system has unique benefits and drawbacks, ultimately, organoids hold special promise for the development of personalized medicine approaches.

Organoid models of human and mouse ductal pancreatic cancer.

Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.

The material convoy after age 50.

OBJECTIVES: Possessions constitute a dynamic "material convoy" that accumulates across adulthood to furnish role enactments and the development of the self. Following a familiar life course arc, older people should hypothetically release the possessions that equipped the daily lives that they no longer have. METHOD: We use new survey data on possession divestment from the 2010 Health and Retirement Study to assess activity on behalf of the material convoy after age 50. RESULTS: After age 50, people are progressively less likely to divest themselves of belongings. After age 70, about 30% of persons say that they have done nothing in the past year to clean out, give away, or donate things, and over 80% have sold nothing. We tested whether divestments diminish with age because they do not seem necessary or because of health limitations, but the age pattern is robust, suggesting retention of the material convoy in later life. DISCUSSION: Further research on this age pattern might consider housing, the construction of the self, and social networks as explanations for retention. Inertia toward the material convoy maintains the availability and comfort of things, but it may also lead to a predicament wherein the collection becomes a worry for self and others.

The yeast Snt2 protein coordinates the transcriptional response to hydrogen peroxide-mediated oxidative stress.

Regulation of gene expression is a vital part of the cellular stress response, yet the full set of proteins that orchestrate this regulation remains unknown. Snt2 is a Saccharomyces cerevisiae protein whose function has not been well characterized that was recently shown to associate with Ecm5 and the Rpd3 deacetylase. Here, we confirm that Snt2, Ecm5, and Rpd3 physically associate. We then demonstrate that cells lacking Rpd3 or Snt2 are resistant to hydrogen peroxide (H2O2)-mediated oxidative stress and use chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to show that Snt2 and Ecm5 recruit Rpd3 to a small number of promoters and in response to H2O2, colocalize independently of Rpd3 to the promoters of stress response genes. By integrating ChIP-seq and expression analyses, we identify target genes that require Snt2 for proper expression after H2O2. Finally, we show that cells lacking Snt2 are also resistant to nutrient stress imparted by the TOR (target of rapamycin) pathway inhibitor rapamycin and identify a common set of genes targeted by Snt2 and Ecm5 in response to both H2O2 and rapamycin. Our results establish a function for Snt2 in regulating transcription in response to oxidative stress and suggest Snt2 may also function in multiple stress pathways.

Oral health problems and mortality.

BACKGROUND/PURPOSE: Previous studies have shown the relationship between individual oral health conditions and mortality; however, the relationship between mortality and multiple oral health conditions has not been examined. This study investigates the link between individual oral health problems and oral comorbidity and mortality risk. MATERIALS AND METHODS: Data are derived from the National Health and Nutrition Examination Survey 1999-2004, which is linked to the National Death Index for mortality follow-up through 2006. We estimated the risk of mortality among people with three individual oral health conditions-tooth loss, root caries, and periodontitis as well as with oral comorbidity-or having all three conditions. RESULTS: Significant tooth loss, root caries, and periodontal disease were associated with increased odds of dying. The relationship between oral health conditions and mortality disappeared when controlling for sociodemographic, health, and/or health behavioral indicators. Having multiple oral health problems was associated with an even higher rate of mortality. CONCLUSION: Individual oral health conditions-tooth loss, root caries, and periodontal disease-were not related to mortality when sociodemographic, health, and/or health behavioral factors were considered, and there was no differential pattern between the three conditions. Multiple oral health problems were associated with a higher risk of dying.

Prevalence of oral health problems in U.S. adults, NHANES 1999-2004: exploring differences by age, education, and race/ethnicity.

Using the National Health and Nutrition Examination Surveys (NHANES) 1999-2004, the authors examined age patterns in oral health indicators by race/ethnicity and socioeconomic status related to edentulism, presence of root caries, and periodontal disease. Our analysis included subjects who were non-Hispanic White, Mexican American, and African American over the age of 20, and who participated in the NHANES oral health examination. African Americans experienced more oral health problems at younger ages; as age increased, so did racial disparities in oral health problems. Lower educational attainment was strongly associated with more oral health problems at all ages. These results may indicate a faster progression of oral health problems with age among African Americans, thus suggesting that the "earlier aging" of members of racial/ethnic minorities which has been reported in prior research may also be found in oral health.

Who are the Latino baby boomers? Demographic and economic characteristics of a hidden population.

The United States is confronting two simultaneous demographic shifts with profound implications for public policy: population aging and increasing diversity. These changes are accelerating during a dramatic economic downturn, placing entitlement reform prominently on the national policy agenda. Using decennial census data from 2000, this paper examines the nexus of these trends by examining characteristics of Latino baby boomers. In the census data, Latinos constituted 10% of the 80 million boomers; roughly one-third of Latino boomers (37%) were born in the United States or abroad to a U.S. parent; 6% were born in a U.S. territory; 21% were naturalized citizens; and 36% were noncitizens. Compared to non-Latinos, Latino baby boomers had lower levels of education, home ownership, and investment income and higher rates of material hardship and poverty; however, there was considerable variation based on citizenship status. A better understanding of Latino baby boomers will help policy makers anticipate the retirement needs of baby boomers as the United States prepares for the aging of a racially and ethnically diverse population.

Poverty and Material Hardship in Grandparent-Headed Households.

Using the 2001 Survey of Income and Program Participation, the current study examines poverty and material hardship among children living in 3-generation (n = 486), skipped-generation (n = 238), single-parent (n = 2,076), and 2-parent (n = 6,061) households. Multinomial and logistic regression models indicated that children living in grandparent-headed households experience elevated risk of health insecurity (as measured by receipt of public insurance and uninsurance)-a disproportionate risk given rates of poverty within those households. Children living with single parents did not share this substantial risk. Risk of food and housing insecurity did not differ significantly from 2-parent households once characteristics of the household and caregivers were taken into account.

The Implications of Grandparent Coresidence for Economic Hardship among Children in Mother-Only Families.

Estimates suggest that more than 6 million children live with at least one grandparent. Despite evidence establishing the growing prevalence of this arrangement, limited research has focused on estimating the implications of co-residence for the economic well-being of grandchildren. Using data from the 2001 panel of the Survey of Income and Program Participation, this paper examines levels of financial hardship among a particularly vulnerable group of children - those living in mother-only families. Findings suggest that children living in mother-only families that include a grandparent are substantially less likely to be living below or near the poverty level, compared to children living in mother-only families without a grandparent present. The financial security of children in these three-generation households is enhanced through significant economic contributions of the grandparents, and from household receipt of a wide range of financial resources, including means-tested cash transfers and other income such as Social Security.

Preventive health behaviors among grandmothers raising grandchildren.

OBJECTIVES: We examined differential preventive health behavior among grandmothers who recently began raising a grandchild, grandmothers raising a grandchild for at least 2 years, and grandmothers not raising a grandchild. METHODS: Data came from the 2000, 2002, and 2004 waves of the Health and Retirement Study. We ran multivariate logistic regression models to assess receipt of influenza vaccination, cholesterol screening, monthly breast self-exam, mammography, and Papanicolaou (Pap) tests among grandmothers aged 50 to 75. RESULTS: Grandmothers who recently began raising a grandchild were significantly less likely to report influenza vaccination and cholesterol screening than grandmothers not raising grandchildren, even after we controlled for increased emotional and financial strains within the household. We also observed this association for Pap tests, although this finding was only marginally significant. Grandmothers who had been raising a grandchild for at least 2 years were significantly more likely to report influenza vaccination and monthly breast self-exam than grandmothers not raising grandchildren. DISCUSSION: The enhancement of preventive behavior seen among long-term grandparent caregivers does not fully offset the suppression of preventive behavior during the transition into care; support groups should target a range of interventions toward the promotion of healthy behavior among new grandparent caregivers.

PHD fingers in human diseases: disorders arising from misinterpreting epigenetic marks.

Histone covalent modifications regulate many, if not all, DNA-templated processes, including gene expression and DNA damage response. The biological consequences of histone modifications are mediated partially by evolutionarily conserved "reader/effector" modules that bind to histone marks in a modification- and context-specific fashion and subsequently enact chromatin changes or recruit other proteins to do so. Recently, the Plant Homeodomain (PHD) finger has emerged as a class of specialized "reader" modules that, in some instances, recognize the methylation status of histone lysine residues, such as histone H3 lysine 4 (H3K4). While mutations in catalytic enzymes that mediate the addition or removal of histone modifications (i.e., "writers" and "erasers") are already known to be involved in various human diseases, mutations in the modification-specific "reader" proteins are only beginning to be recognized as contributing to human diseases. For instance, point mutations, deletions or chromosomal translocations that target PHD fingers encoded by many genes (such as recombination activating gene 2 (RAG2), Inhibitor of Growth (ING), nuclear receptor-binding SET domain-containing 1 (NSD1) and Alpha Thalassaemia and Mental Retardation Syndrome, X-linked (ATRX)) have been associated with a wide range of human pathologies including immunological disorders, cancers, and neurological diseases. In this review, we will discuss the structural features of PHD fingers as well as the diseases for which direct mutation or dysregulation of the PHD finger has been reported. We propose that misinterpretation of the epigenetic marks may serve as a general mechanism for human diseases of this category. Determining the regulatory roles of histone covalent modifications in the context of human disease will allow for a more thorough understanding of normal and pathological development, and may provide innovative therapeutic strategies wherein "chromatin readers" stand as potential drug targets.