Loss of PPARγ activity characterizes early protumorigenic stromal reprogramming and dictates the therapeutic window of opportunity.

Although robustly expressed in the disease-free (DF) breast stroma, CD36 is consistently absent from the stroma surrounding invasive breast cancers (IBCs). In this study, we primarily observed CD36 expression in adipocytes and intralobular capillaries within the DF breast. Larger vessels concentrated in interlobular regions lacked CD36 and were instead marked by the expression of CD31. When evaluated in perilesional capillaries surrounding ductal carcinoma in situ, a nonobligate IBC precursor, CD36 loss was more commonly observed in lesions associated with subsequent IBC. Peroxisome proliferator-activated receptor γ (PPARγ) governs the expression of CD36 and genes involved in differentiation, metabolism, angiogenesis, and inflammation. Coincident with CD36 loss, we observed a dramatic suppression of PPARγ and its target genes in capillary endothelial cells (ECs) and pericytes, which typically surround and support the stability of the capillary endothelium. Factors present in conditioned media from malignant cells repressed PPARγ and its target genes not only in cultured ECs and pericytes but also in adipocytes, which require PPARγ for proper differentiation. In addition, we identified a role for PPARγ in opposing the transition of pericytes toward a tumor-supportive myofibroblast phenotype. In mouse xenograft models, early intervention with rosiglitazone, a PPARγ agonist, demonstrated significant antitumor effects; however, following the development of a palpable tumor, the antitumor effects of rosiglitazone were negated by the repression of PPARγ in the mouse stroma. In summary, PPARγ activity in healthy tissues places several stromal cell types in an antitumorigenic state, directly inhibiting EC proliferation, maintaining adipocyte differentiation, and suppressing the transition of pericytes into tumor-supportive myofibroblasts.

Concerted epithelial and stromal changes during progression of Barrett's Esophagus to invasive adenocarcinoma exposed by multi-scale, multi-omics analysis.

Esophageal adenocarcinoma arises from Barrett's esophagus, a precancerous metaplastic replacement of squamous by columnar epithelium in response to chronic inflammation. Multi-omics profiling, integrating single-cell transcriptomics, extracellular matrix proteomics, tissue-mechanics and spatial proteomics of 64 samples from 12 patients' paths of progression from squamous epithelium through metaplasia, dysplasia to adenocarcinoma, revealed shared and patient-specific progression characteristics. The classic metaplastic replacement of epithelial cells was paralleled by metaplastic changes in stromal cells, ECM and tissue stiffness. Strikingly, this change in tissue state at metaplasia was already accompanied by appearance of fibroblasts with characteristics of carcinoma-associated fibroblasts and of an NK cell-associated immunosuppressive microenvironment. Thus, Barrett's esophagus progresses as a coordinated multi-component system, supporting treatment paradigms that go beyond targeting cancerous cells to incorporating stromal reprogramming.

Apposition of Fibroblasts With Metaplastic Gastric Cells Promotes Dysplastic Transition.

BACKGROUND & AIMS: Elements of field cancerization, including atrophic gastritis, metaplasia, and dysplasia, promote gastric cancer development in association with chronic inflammation. However, it remains unclear how stroma changes during carcinogenesis and how the stroma contributes to progression of gastric preneoplasia. Here we investigated heterogeneity of fibroblasts, one of the most important elements in the stroma, and their roles in neoplastic transformation of metaplasia. METHODS: We used single-cell transcriptomics to evaluate the cellular heterogeneity of mucosal cells from patients with gastric cancer. Tissue sections from the same cohort and tissue microarrays were used to identify the geographical distribution of distinct fibroblast subsets. We further evaluated the role of fibroblasts from pathologic mucosa in dysplastic progression of metaplastic cells using patient-derived metaplastic gastroids and fibroblasts. RESULTS: We identified 4 subsets of fibroblasts within stromal cells defined by the differential expression of PDGFRA, FBLN2, ACTA2, or PDGFRB. Each subset was distributed distinctively throughout stomach tissues with different proportions at each pathologic stage. The PDGFRα+ subset expanded in metaplasia and cancer compared with normal, maintaining a close proximity with the epithelial compartment. Co-culture of metaplasia- or cancer-derived fibroblasts with gastroids showing the characteristics of spasmolytic polypeptide-expressing metaplasia-induced disordered growth, loss of metaplastic markers, and increases in markers of dysplasia. Culture of metaplastic gastroids with conditioned media from metaplasia- or cancer-derived fibroblasts also promoted dysplastic transition. CONCLUSIONS: These findings indicate that fibroblast associations with metaplastic epithelial cells can facilitate direct transition of metaplastic spasmolytic polypeptide-expressing metaplasia cell lineages into dysplastic lineages.

Data-independent acquisition and quantification of extracellular matrix from human lung in chronic inflammation-associated carcinomas.

Early events associated with chronic inflammation and cancer involve significant remodeling of the extracellular matrix (ECM), which greatly affects its composition and functional properties. Using lung squamous cell carcinoma (LSCC), a chronic inflammation-associated cancer (CIAC), we optimized a robust proteomic pipeline to discover potential biomarker signatures and protein changes specifically in the stroma. We combined ECM enrichment from fresh human tissues, data-independent acquisition (DIA) strategies, and stringent statistical processing to analyze "Tumor" and matched adjacent histologically normal ("Matched Normal") tissues from patients with LSCC. Overall, 1802 protein groups were quantified with at least two unique peptides, and 56% of those proteins were annotated as "extracellular." Confirming dramatic ECM remodeling during CIAC progression, 529 proteins were significantly altered in the "Tumor" compared to "Matched Normal" tissues. The signature was typified by a coordinated loss of basement membrane proteins and small leucine-rich proteins. The dramatic increase in the stromal levels of SERPINH1/heat shock protein 47, that was discovered using our ECM proteomic pipeline, was validated by immunohistochemistry (IHC) of "Tumor" and "Matched Normal" tissues, obtained from an independent cohort of LSCC patients. This integrated workflow provided novel insights into ECM remodeling during CIAC progression, and identified potential biomarker signatures and future therapeutic targets.

Antigen-Dependent Inducible T-Cell Reporter System for PET Imaging of Breast Cancer and Glioblastoma.

For the past several decades, chimeric antigen receptor T-cell therapies have shown promise in the treatment of cancers. These treatments would greatly benefit from companion imaging biomarkers to follow the trafficking of T cells in vivo. Methods: Using synthetic biology, we engineered T cells with a chimeric receptor synthetic intramembrane proteolysis receptor (SNIPR) that induces overexpression of an exogenous reporter gene cassette on recognition of specific tumor markers. We then applied a SNIPR-based PET reporter system to 2 cancer-relevant antigens, human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor variant III (EGFRvIII), commonly expressed in breast and glial tumors, respectively. Results: Antigen-specific reporter induction of the SNIPR PET T cells was confirmed in vitro using green fluorescent protein fluorescence, luciferase luminescence, and the HSV-TK PET reporter with 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine ([18F]FHBG). T cells associated with their target antigens were successfully imaged using PET in dual-xenograft HER2+/HER2- and EGFRvIII+/EGFRvIII- animal models, with more than 10-fold higher [18F]FHBG signals seen in antigen-expressing tumors versus the corresponding controls. Conclusion: The main innovation found in this work was PET detection of T cells via specific antigen-induced signals, in contrast to reporter systems relying on constitutive gene expression.

Cost Effectiveness and Budget Impact of the Boston University Approach to Psychiatric Rehabilitation for Increasing the Social Participation of Individuals With Severe Mental Illnesses.

Background: The purpose of this study was to investigate the cost-effectiveness and budget impact of the Boston University Approach to Psychiatric Rehabilitation (BPR) compared to an active control condition (ACC) to increase the social participation (in competitive employment, unpaid work, education, and meaningful daily activities) of individuals with severe mental illnesses (SMIs). ACC can be described as treatment as usual but with an active component, namely the explicit assignment of providing support with rehabilitation goals in the area of social participation. Method: In a randomized clinical trial with 188 individuals with SMIs, BPR (n = 98) was compared to ACC (n = 90). Costs were assessed with the Treatment Inventory of Costs in Patients with psychiatric disorders (TIC-P). Outcome measures for the cost-effectiveness analysis were incremental cost per Quality Adjusted Life Year (QALY) and incremental cost per proportional change in social participation. Budget Impact was investigated using four implementation scenarios and two costing variants. Results: Total costs per participant at 12-month follow-up were € 12,886 in BPR and € 12,012 in ACC, a non-significant difference. There were no differences with regard to social participation or QALYs. Therefore, BPR was not cost-effective compared to ACC. Types of expenditure with the highest costs were in order of magnitude: supported and sheltered housing, inpatient care, outpatient care, and organized activities. Estimated budget impact of wide BPR implementation ranged from cost savings to €190 million, depending on assumptions regarding uptake. There were no differences between the two costing variants meaning that from a health insurer perspective, there would be no additional costs if BPR was implemented on a wider scale in mental health care institutions. Conclusions: This was the first study to investigate BPR cost-effectiveness and budget impact. The results showed that BPR was not cost-effective compared to ACC. When interpreting the results, one must keep in mind that the cost-effectiveness of BPR was investigated in the area of social participation, while BPR was designed to offer support in all rehabilitation areas. Therefore, more studies are needed before definite conclusions can be drawn on the cost-effectiveness of the method as a whole.

Students' Perceptions of the Integration of Sexual and Gender Minority Constructs within the Dental Curriculum

Background and Objective@#The sexual and gender minorities (SGMs) are at risk for various health disparities. To address this, the World Health Organization (WHO) calls for the training of healthcare providers who demonstrate cultural competence. To facilitate this, studies have suggested the inclusion of SGM-specific constructs in the curriculum that is complemented with campus climate-related efforts. This study aimed to determine the degree of coverage of cultural competence towards the SGM constructs in the required curriculum and describe the campus climate towards the SGM of a dental school in the Philippines from the students' perspective. @*Methodology@#A convergent parallel mixed-methods design was employed. A survey questionnaire was developed and administered to determine the coverage of SGM-specific constructs. This was followed by semi-structured interviews to discuss the campus climate towards the SGM. Data collection utilized a twostage stratified random sampling method for the survey while a purposive sampling method was used for interviewing dental students enrolled in a private, non-sectarian school. @*Results and Conclusion@#Survey results show that the most reported covered concepts are the oral manifestations of sexually transmitted diseases and the use of inclusive terminologies. Interview responses reveal instances of inadvertent perpetuation of heteronormative ideologies, however, the campus climate is perceived as neutral towards the SGM. The integration of the two datasets reveals incongruences between the curricular coverage and the perceived campus climate. Nevertheless, findings imply that the dental curriculum remains largely objective and asexual. Additionally, the integration underscores the importance of complementing curricular content with an inclusive campus climate in developing cultural competence among dental students.

Spinal Cord Ischemia Related to Disc Herniation: Case Report and a Review of the Literature.

Symptoms of spinal cord ischemia can mimic myelopathy due to spinal cord compression in the acute phase. Thoracic disc herniation with limited spinal cord compression but rapid progression of neurological symptoms causes a clinical dilemma as to whether emergency decompression should be performed. We report a case of acute progressive myelopathy due to spinal cord ischemia related to thoracic disc herniation initially managed by Th8 laminectomy with reduction of the herniated disc. Repeat imaging showed T2-weighted hyperintensity in the posterior cord. The clinical and radiological course supports posterior spinal artery ischemia. This case illustrates and a review of the literature shows that thoracic disc herniation may be complicated by ischemic myelopathy even in the absence of cord compression.

Asymptomatic Bordetella pertussis infections in young African infants and their mothers identified within a longitudinal cohort.

Despite long-standing vaccination programs, pertussis incidence has increased in numerous countries; transmission by asymptomatic individuals is a suspected driver of this resurgence. However, unequivocal evidence documenting asymptomatic infections in adults and children is lacking due, in part, to the cross-sectional nature of most pertussis surveillance studies. In addition, modern pertussis surveillance relies on quantitative PCR (qPCR) using fixed diagnostic thresholds to identify cases. To address this gap, we present a longitudinal analysis of 17,442 nasopharyngeal samples collected from a cohort of 1,320 Zambian mother/infant pairs. Using full-range cycle threshold (CT) values from IS481 qPCR assays, we document widespread asymptomatic infections among mothers and also, surprisingly, among young infants. From an initial group of eight symptomatic infants who tested positive by qPCR, we identify frequent contemporaneous subclinical infections in mothers. Within the full cohort, we observe strong temporal correlation between low- and high-intensity qPCR signals. We compute a single time-averaged score for each individual summarizing the evidence for pertussis infection (EFI), and show that EFI strongly clusters within mother/infant pairs, and is strongly associated with clinical symptomatology and antibiotic use. Overall, the burden of pertussis here is substantially underestimated when restricting diagnostic criteria to IS481 CT≤35. Rather, we find that full-range CT values provide valuable insights into pertussis epidemiology in this population, and illuminate the infection arc within individuals. These findings have significant implications for quantifying asymptomatic pertussis prevalence and its contribution to overall transmission. Our results also expose limitations of threshold-based interpretations of qPCR assays in infectious disease surveillance.

A framework for advancing our understanding of cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) are a key component of the tumour microenvironment with diverse functions, including matrix deposition and remodelling, extensive reciprocal signalling interactions with cancer cells and crosstalk with infiltrating leukocytes. As such, they are a potential target for optimizing therapeutic strategies against cancer. However, many challenges are present in ongoing attempts to modulate CAFs for therapeutic benefit. These include limitations in our understanding of the origin of CAFs and heterogeneity in CAF function, with it being desirable to retain some antitumorigenic functions. On the basis of a meeting of experts in the field of CAF biology, we summarize in this Consensus Statement our current knowledge and present a framework for advancing our understanding of this critical cell type within the tumour microenvironment.

Metastatic State of Cancer Cells May Be Indicated by Adhesion Strength.

Cancer cells within a tumor are heterogeneous and only a small fraction are able to form secondary tumors. Universal biological markers that clearly identify potentially metastatic cells are limited, which complicates isolation and further study. However, using physical rather than biological characteristics, we have identified Mg2+- and Ca2+-mediated differences in adhesion strength between metastatic and nonmetastatic mammary epithelial cell lines, which occur over concentration ranges similar to those found in tumor stroma. Metastatic cells exhibit remarkable heterogeneity in their adhesion strength under stromal-like conditions, unlike their nonmetastatic counterparts, which exhibit Mg2+- and Ca2+-insensitive adhesion. This heterogeneity is the result of increased sensitivity to Mg2+- and Ca2+-mediated focal adhesion disassembly in metastatic cells, rather than changes in integrin expression or focal adhesion phosphorylation. Strongly adherent metastatic cells exhibit less migratory behavior, similar to nonmetastatic cell lines but contrary to the unselected metastatic cell population. Adhesion strength heterogeneity was observed across multiple cancer cell lines as well as isogenically, suggesting that adhesion strength may serve as a general marker of metastatic cells.

SOX2, OCT3/4 and NANOG expression and cellular plasticity in rare human somatic cells requires CD73.

Endogenous Plastic Somatic (ePS) cells isolated from adult human tissues exhibit extensive lineage plasticity in vitro and in vivo. Here we visualize these rare ePS cells in a latent state, i.e. lacking SOX2, OCT3/4 and NANOG (SON) expression, in non-diseased breast specimens through immunohistochemical analysis of previously identified ePS-specific biomarkers (CD73+, EpCAM+ and CD90-). We also report a novel mechanism by which these latent ePS cells acquire SON expression and plasticity in vitro. Four extracellular factors are necessary for the acquisition of SON expression and lineage plasticity in ePS cells: adenosine (which is produced by the 5' ecto-nucleotidase CD73 and activates in turn the PKA-dependent IL6/STAT3 pathway through the adenosine receptor ADORA2b), IL6, FGF2 and ACTIVIN A. Blocking any pathway component renders ePS cells incapable of SON expression and lineage plasticity. Notably, hESCs do not use adenosine or IL6 nor they express CD73 or ADORA2b and inhibition of adenosine signaling does not ablate their plasticity. Therefore, the data presented here delineate novel circuitry and physiological signals for accessing SON expression in rare, undifferentiated human cells.

Carcinoma-associated fibroblasts: orchestrating the composition of malignancy.

The tumor stroma is no longer seen solely as physical support for mutated epithelial cells but as an important modulator and even a driver of tumorigenicity. Within the tumor stromal milieu, heterogeneous populations of fibroblast-like cells, collectively termed carcinoma-associated fibroblasts (CAFs), are key players in the multicellular, stromal-dependent alterations that contribute to malignant initiation and progression. This review focuses on novel insights into the contributions of CAFs to disease progression, emergent events leading to the generation of CAFs, identification of CAF-specific biomarkers predictive of disease outcome, and recent therapeutic approaches aimed at blunting or reverting detrimental protumorigenic phenotypes associated with CAFs.

Risk prediction for local versus regional/metastatic tumors after initial ductal carcinoma in situ diagnosis treated by lumpectomy.

Among women diagnosed with ductal carcinoma in situ (DCIS), we identified factors associated with local invasive cancer (LIC) and regional/metastatic invasive cancer (RMIC) and provide 10-year risks based on clinically relevant factors. We created a retrospective, population-based cohort of 1492 women with an initial diagnosis of DCIS (1983-1996) treated by lumpectomy alone. Histological and molecular markers (Ki67, ER, PR, COX-2, p16, ERBB2) were collected on DCIS cases with a subsequent tumor (DCIS, LIC, or RMIC) and a subsample of frequency-matched controls without subsequent tumors. Competing risks methods were used to identify factors associated with LIC and RMIC and cumulative incidence methods to estimate 10-year risks for combinations of factors. Median follow-up time was 12.6 years (range 0.5-29.5 years). The overall 10-year risk of LIC (11.9 %) was higher than for RMIC (3.8 %). About half of women with initial DCIS lesions are detected by mammography and p16 negative and have a 10-year risk of LIC of 6.2 % (95 % CI 5.8-6.8 %) and RMIC of 1.2 % (95 % CI 1.1-1.3 %). Premenopausal women whose DCIS lesion was p16 positive or p16 negative and detected by palpation had high 10-year risk of LIC of 23.0 % (95 % CI 19.3-27.4 %). Ten-year risk of RMIC was highest at 22.5 % (95 % CI 13.8-48.1 %) for those positive for p16, COX-2, and ERRB2, and negative for ER, but prevalence of this group is low at 3 %. Ten-year risk of LIC and RMIC is low for the majority diagnosed with DCIS. Combinations of molecular markers and method of detection of initial DCIS lesion can differentiate women at low and high risk of LIC and RMIC.

Novel insights from 3D models: the pivotal role of physical symmetry in epithelial organization.

3D tissue culture models are utilized to study breast cancer and other pathologies because they better capture the complexity of in vivo tissue architecture compared to 2D models. However, to mimic the in vivo environment, the mechanics and geometry of the ECM must also be considered. Here, we studied the mechanical environment created in two 3D models, the overlay protocol (OP) and embedded protocol (EP). Mammary epithelial acini features were compared using OP or EP under conditions known to alter acinus organization, i.e. collagen crosslinking and/or ErbB2 receptor activation. Finite element analysis and active microrheology demonstrated that OP creates a physically asymmetric environment with non-uniform mechanical stresses in radial and circumferential directions. Further contrasting with EP, acini in OP displayed cooperation between ErbB2 signalling and matrix crosslinking. These differences in acini phenotype observed between OP and EP highlight the functional impact of physical symmetry in 3D tissue culture models.

Towards aspirin-inspired self-immolating molecules which target the cyclooxygenases.

Cyclooxygenases (COXs) are enzymes that play a vital role in the inflammatory cascade through the generation of prostaglandins. Their over-expression has been implicated in numerous diseases. In particular, over-expression of COX-2 has been shown to be a predictive biomarker for progression of pre-malignant lesions towards invasive cancer in various tissues. This makes the early detection of COX-2 expressing lesions of high clinical relevance. Herein we describe the development of the first self-immolating trigger which targets COXs. We incorporated our trigger design into 2 activatable fluorogenic probes and demonstrated COX-specific activation in vitro. Experimental data revealed probe activation was likely caused by solvent-exposed amino acids on the surface of the COXs. Overall, the probes reported here mark the first step towards developing self-immolating imaging/therapeutic agents targeted to specific COXs.

Epigenetic and transcriptional determinants of the human breast.

While significant effort has been dedicated to the characterization of epigenetic changes associated with prenatal differentiation, relatively little is known about the epigenetic changes that accompany post-natal differentiation where fully functional differentiated cell types with limited lifespans arise. Here we sought to address this gap by generating epigenomic and transcriptional profiles from primary human breast cell types isolated from disease-free human subjects. From these data we define a comprehensive human breast transcriptional network, including a set of myoepithelial- and luminal epithelial-specific intronic retention events. Intersection of epigenetic states with RNA expression from distinct breast epithelium lineages demonstrates that mCpG provides a stable record of exonic and intronic usage, whereas H3K36me3 is dynamic. We find a striking asymmetry in epigenomic reprogramming between luminal and myoepithelial cell types, with the genomes of luminal cells harbouring more than twice the number of hypomethylated enhancer elements compared with myoepithelial cells.