Evidence for EpCAM and Cytokeratin Expressing Epithelial Cells in Normal Human and Murine Blood and Bone Marrow.

Epithelial cells have been identified in the blood and bone marrow of patients with cancer and other diseases. However, the presence of normal epithelial cells in the blood and bone marrow of healthy individuals has yet to be identified in a consistent way. Presented here is a reproducible method for isolating epithelial cells from healthy human and murine blood and bone marrow (BM) using flow cytometry and immunofluorescence (IF) microscopy. Epithelial cells in healthy individuals were first identified and isolated via flow cytometry using epithelial cell adhesion molecule (EpCAM). These EpCAM+ cells were confirmed to express keratin using immunofluorescence microscopy in Krt1-14;mTmG transgenic mice. Human blood samples had 0.18% ± 0.0004 EpCAM+ cells (SEM; n=7 biological replicates, 4 experimental replicates). In human BM, 3.53% ± 0.006 (SEM; n=3 biological replicates, 4 experimental replicates) of mononuclear cells were EpCAM+. In mouse blood, EpCAM+ cells constituted 0.45% ± 0.0006 (SEM; n=2 biological replicates, 4 experimental replicates), and in mouse BM, 5.17% ± 0.001 (SEM; n=3 biological replicates, 4 experimental replicates) were EpCAM+. In mice, all the EpCAM+ cells were immunoreactive to pan-cytokeratin, as determined by IF microscopy. Results were confirmed using Krt1-14;mTmG transgenic mice, with low (8.6 native GFP+ cells per 106 cells analyzed; 0.085% of viable cells), but significant numbers (p < 0.0005) of GFP+ cells present in normal murine BM, that were not the result of randomness compared with multiple negative controls. Further, EpCAM+ cells in mouse blood were more heterogeneous than CD45+ cells (0.58% in BM; 0.13% in blood). These observations conclude that cells expressing cytokeratin proteins are reproducibly detectable among mononuclear cells from human and murine blood and BM. We demonstrate a method of tissue harvesting, flow cytometry, and immunostaining that can be used to identify and determine the function of these pan-cytokeratin epithelial cells in healthy individuals.

Buried island transposition flap for joint Plastic-Neurosurgical management of spinal wound dehiscence - a technical note and single Centre experience.

INTRODUCTION: Spinal surgical wound infection can lead to tissue voids between the spine and skin that can be difficult to reconstruct. Previously described techniques include myocutaneous flaps or perforator based fasciocutaneous flaps. However, these procedures can be time-consuming and surgically challenging. AIMS: This study aimed to assess the effectiveness of a novel technique employing a buried island transposition (BIT) flap, for the repair of non-irradiated dehisced spinal wounds. METHODS: Fifteen patients with failed conservative management of infected midline posterior spinal wounds, underwent wound repair using a local buried islanded de-epithelialized double-breasted fasciocutaneous transposition flap, performed by joint input from the neurosurgical and plastic surgical teams. RESULTS: Mean age was 58 years (range, 31-76 years) with male-to-female ratio of 8:7. The BIT flap was used to repair four wounds in the cervical spine with underlying fixation; four wounds in the thoracic spine with underlying fixation; and seven wounds in the lumbar-sacral spine, of which three had underlying fixation. Pre-operatively, each of the wounds were either dehiscent with exposed hardware, or had large defects unsuitable for primary closure following debridement. There was no procedure-related mortality. All patients demonstrated good wound healing with no subsequent repeat surgery or removal of spinal fixation at mean 24-month follow-up. CONCLUSION: We successfully used a novel buried island transposition flap that has not previously been described in repair of spinal wounds. This technique, which led in all cases to good wound healing and prevented removal of metalwork, has comparable efficacy but increased ease of use compared to traditional techniques. It requires redundant skin at the wound site.

Ionizing Radiation-Induced DNA Damage Response in Primary Melanocytes and Keratinocytes of Human Skin.

Currently, our knowledge of how different cell types in a tissue microenvironment respond to low and high linear energy transfer (LET) radiation is highly restricted. In this study, a comparative analysis was performed on γ-ray-induced DNA damage and repair in primary human melanocytes and keratinocytes isolated from 3 donors. Our study demonstrates a modest interindividual variability in both melanocytes and keratinocytes in terms of both spontaneous and ionizing radiation (IR)-induced 53BP1 foci formation and persistence. Melanocytes, in general, showed a slightly elevated (1.66-2.79 folds more) 53BP1 foci induction relative to keratinocytes after exposure to different doses of γ-rays (0.1-2.5 Gy) radiation. To verify the influence of ATM kinase on IR-induced 53BP1 foci formation, melanocytes and keratinocytes were treated with a specific ATM kinase inhibitor (KU55993, 10 µM) for 1 h prior to radiation. ATM kinase inhibition resulted in the reduction of both spontaneous and IR-induced 53BP1 foci by 17-42% in both melanocytes and keratinocytes of all the 3 donors. Increased persistence of IR-induced 53BP1 foci number was observed in ATM-inhibited melanocytes and keratinocytes after different post exposure times (6 h and 24 h). Taken together, our study suggests that interindividual variations exist in the induction and repair of DNA double-strand breaks (DSBs) in melanocytes and keratinocytes and that ATM is crucial for an optimal DSB repair efficiency in both human skin cell types.

Prostaglandin Pathways: Opportunities for Cancer Prevention and Therapy.

Because of profound effects observed in carcinogenesis, prostaglandins (PG), prostaglandin-endoperoxide synthases, and PG receptors are implicated in cancer development and progression. Understanding the molecular mechanisms of PG actions has potential clinical relevance for cancer prevention and therapy. This review focuses on the current status of PG signaling pathways in modulating cancer progression and aims to provide insights into the mechanistic actions of PGs and their receptors in influencing tumor progression. We also examine several small molecules identified as having anticancer activity that target prostaglandin receptors. The literature suggests that targeting PG pathways could provide opportunities for cancer prevention and therapy.

Telerehabilitation's Safety, Feasibility, and Exercise Uptake in Cancer Survivors: Process Evaluation.

BACKGROUND: Access to exercise for cancer survivors is poor despite global recognition of its benefits. Telerehabilitation may overcome barriers to exercise for cancer survivors but is not routinely offered. OBJECTIVE: Following the rapid implementation of an exercise-based telerehabilitation program in response to COVID-19, a process evaluation was conducted to understand the impact on patients, staff, and the health service with the aim of informing future program development. METHODS: A mixed methods evaluation was completed for a telerehabilitation program for cancer survivors admitted between March and December 2020. Interviews were conducted with patients and staff involved in implementation. Routinely collected hospital data (adverse events, referrals, admissions, wait time, attendance, physical activity, and quality of life) were also assessed. Patients received an 8-week telerehabilitation intervention including one-on-one health coaching via telehealth, online group exercise and education, information portal, and home exercise prescription. Quantitative data were reported descriptively, and qualitative interview data were coded and mapped to the Proctor model for implementation research. RESULTS: The telerehabilitation program received 175 new referrals over 8 months. Of those eligible, 123 of 150 (82%) commenced the study. There were no major adverse events. Adherence to health coaching was high (674/843, 80% of scheduled sessions), but participation in online group exercise classes was low (n=36, 29%). Patients improved their self-reported physical activity levels by a median of 110 minutes per week (IQR 90-401) by program completion. Patients were satisfied with telerehabilitation, but clinicians reported a mixed experience of pride in rapid care delivery contrasting with loss of personal connections. The average health service cost per patient was Aus $1104 (US $790). CONCLUSIONS: Telerehabilitation is safe, feasible, and improved outcomes for cancer survivors. Learnings from this study may inform the ongoing implementation of cancer telerehabilitation.

Patient priorities in relation to surgery for gastric cancer: qualitative interviews with gastric cancer surgery patients to inform the development of a core outcome set.

OBJECTIVE: The reporting of outcomes in surgical trials for gastric cancer is inconsistent. The GASTROS study (GAstric Cancer Surgery TRials Reported Outcome Standardisation) aims to address this by developing a core outcome set (COS) for use in all future trials within this field. A COS should reflect the views of all stakeholders, including patients. We undertook a series of interviews to identify outcomes important to patients which would be considered for inclusion in a COS. SETTING: All interviews took place within the UK. Interviews were carried out face-to-face at hospitals and cancer support centres or via the telephone. PARTICIPANTS: Twenty participants at varying stages of recovery following surgery for gastric cancer with curative intent. DESIGN: Qualitative design using semistructured interviews, supported by an interview guide which was iteratively modified; thematic analysis was used to explore patient priorities. RESULTS: Six themes enveloping 38 outcomes were identified; surviving and controlling cancer, technical aspects of surgery, adverse events from surgery, recovering from surgery, long-term problems following surgery and long-term life impact of surgery. The 'most important' patient priority was to be 'cured of cancer'. CONCLUSION: Surgical trials for gastric cancer should consider broader priorities of patients when choosing which outcomes to report. This study highlighted the importance of longer-term outcomes such as cancer survival. Outcomes identified in this study will be used to inform an international Delphi survey to develop a COS in this field.

Never events in general practice: a focus group study exploring the views of English and Scottish general practitioners of 'never events'.

OBJECTIVES: To examine general practitioner (GP) understanding of the never event (NE) concept in general practice, and to identify potential enablers and barriers to implementation in UK general practice. DESIGN: Qualitative study using focus groups. The data were analysed thematically and were informed by the normalisation process theory. SETTING: General practice in Northwest England and Southwest Scotland. PARTICIPANTS: 25 GPs took part in five focus groups. 13 GPs were female and 12 male with an age range of 28-60. RESULTS: The NE approach of avoiding serious preventable adverse outcomes from healthcare fitted with participants expectations of the delivery of care but the implementation of strategies to prevent the specific NE was considered complex and variable. The main themes identified participants' understandings and perceived limitations of the NE concept; the embedded layers of responsibility to implement NE within practices and the work required for implementation within general practices. Participants' accounts highlighted the differential nature of work in general practice and that the implementation of initiatives to address specific NE should be situated within a learning and systems approach to implementation. Some NEs were considered more relevant and amenable to simple solutions than others which could influence implementation. CONCLUSIONS: The NE concept was considered overall an important approach to help address key primary care patient safety issues. The utility of individual NEs may vary depending on the complexity of the initiatives that would be needed to manage related risks to as low as reasonably practicable.

Bone marrow-derived epithelial cells and hair follicle stem cells contribute to development of chronic cutaneous neoplasms.

We used allogeneic bone marrow transplantation (BMT) and a mouse multistage cutaneous carcinogenesis model to probe recruitment of bone marrow-derived epithelial cells (BMDECs) in skin tumors initiated with the carcinogen, dimethylbenz[a]anthracene (DMBA), and promoted with 12-O-tetradecanolyphorbol-13-acetate (TPA). BMDECs clustered in the lesional epithelium, expressed cytokeratins, proliferated, and stratified. We detected cytokeratin induction in plastic-adherent bone marrow cells (BMCs) cultured in the presence of filter-separated keratinocytes (KCs) and bone morphogenetic protein 5 (BMP5). Lineage-depleted BMCs migrated towards High Mobility Group Box 1 (HMGB1) protein and epidermal KCs in ex vivo invasion assays. Naive female mice receiving BMTs from DMBA-treated donors developed benign and malignant lesions after TPA promotion alone. We conclude that BMDECs contribute to the development of papillomas and dysplasia, demonstrating a systemic contribution to these lesions. Furthermore, carcinogen-exposed BMCs can initiate benign and malignant lesions upon tumor promotion. Ultimately, these findings may suggest targets for treatment of non-melanoma skin cancers.

Epidermal FABP Prevents Chemical-Induced Skin Tumorigenesis by Regulation of TPA-Induced IFN/p53/SOX2 Pathway in Keratinocytes.

Skin lipids (e.g., fatty acids) are essential for normal skin functions. Epidermal FABP (E-FABP) is the predominant FABP expressed in skin epidermis. However, the role of E-FABP in skin homeostasis and pathology remains largely unknown. Herein, we utilized the 7,12-dimethylbenz(a)anthracene and 12-O-tetradecanolyphorbol-13-acetate-induced skin tumorigenesis model to assess the role of E-FABP in chemical-induced skin tumorigenesis. Compared to their wild-type littermates, mice deficient in E-FABP, but not adipose FABP, developed more skin tumors with higher incidence. 12-O-tetradecanolyphorbol-13-acetate functioning as a tumor promoter induced E-FABP expression and initiated extensive flaring inflammation in skin. Interestingly, 12-O-tetradecanolyphorbol-13-acetate -induced production of IFN-ß and IFN-λ in the skin tissue was dependent on E-FABP expression. Further protein and gene expression arrays demonstrated that E-FABP was critical in enhancing IFN-induced p53 responses and in suppressing SOX2 expression in keratinocytes. Thus, E-FABP expression in skin suppresses chemical-induced skin tumorigenesis through regulation of IFN/p53/SOX2 pathway. Collectively, our data suggest an unknown function of E-FABP in prevention of skin tumor development, and offer E-FABP as a therapeutic target for improving skin innate immunity in chemical-induced skin tumor prevention.

Hmga2 translocation induced in skin tumorigenesis.

Hmga2 protein, a transcription factor involved in chromatin architecture, is expressed chiefly during development, where it has many key biological functions. When expressed in adult tissues from in various organs, Hmga2 is always related to cancer development. The role of Hmga2 in skin tumorigenesis is, however, not yet understood. We demonstrated that Hmga2 can be found in non-transformed epidermis, specifically located to the membrane of keratinocytes (KCs) in epidermis. Ex vivo culture of KCs and development of skin carcinomas in DMBA and TPA mouse models was associated with translocation of the Hmga2 protein from the membrane into the nucleus, where Hmga2 induced its own expression by binding to the Hmga2 promoter. Panobinostat, an HDAC inhibitor, downregulated Hmga2 expression by preventing Hmga2 to bind its own promoter, and thus inhibiting Hmga2 promoter activity. Hmga2 translocation to the nucleus could in part be prevented by an inhibitor for ROCK1. Our findings demonstrate that upon program of benign papilloma to malignant cSCC of skin tumorigenesis, Hmga2 translocates in a ROCK-dependent manner from the membrane to the nucleus, where it serves as an autoregulatory transcription factor, causing cell transformation.

Epidermal Fatty Acid binding protein promotes skin inflammation induced by high-fat diet.

Defining specific cellular and molecular mechanisms in most obesity-related diseases remains an important challenge. Here we report a serendipitous finding that consumption of a high-fat diet (HFD) greatly increased the occurrence of skin lesions in C57BL/6 mice. We demonstrated that HFD induced the accumulation of a specific type of CD11c(+) macrophages in skin preceding detectable lesions. These cells primed skin to induce IL-1ß and IL-18 signaling, which further promoted the cytokines IFN-γ- and IL-17-mediated skin inflammation. Mechanistically, epidermal fatty acid binding protein (E-FABP) was significantly upregulated in skin of obese mice, which coupled lipid droplet formation and NLRP3 inflammasome activation. Deficiency of E-FABP in obese mice decreased recruitment of CD11c(+) macrophages in skin tissues, reduced production of IL-1ß and IL-18, and consequently dampened activation of effector T cells. Furthermore, E-FABP-deficient mice are completely resistant to HFD-induced skin lesions. Collectively, E-FABP represents a molecular sensor triggering HFD-induced skin inflammation.

Participation in voluntary and community organisations in the United Kingdom and the influences on the self-management of long-term conditions.

Voluntary and community organisations (VCOs) have health benefits for those who attend and are viewed as having the potential to support long-term condition management. However, existing community-level understandings of participation do not explain the involvement with VCOs at an individual level, or the nature of support, which may elicit health benefits. Framing active participation as 'doing and experiencing', the aim of this qualitative study was to explore why people with long-term vascular conditions join VCOs, maintain their membership and what prevents participation. Twenty participants, self-diagnosed as having diabetes, chronic heart disease or chronic kidney disease, were purposefully sampled and recruited from a range of VCOs in the North West of England identified from a mapping of local organisations. In semi-structured interviews, we explored the nature of their participation. Analysis was thematic and iterative involving a continual reflection on the data. People gave various reasons for joining groups. These included health and well-being, the need for social contact and pursuing a particular hobby. Barriers to participation included temporal and spatial barriers and those associated with group dynamics. Members maintained their membership on the basis of an identity and sense of belonging to the group, developing close relationships within it and the availability of support and trust. Participants joined community groups often in response to a health-related event. Our findings demonstrate the ways in which the social contact associated with continued participation in VCOs is seen as helping with long-term condition management. Interventions designed at improving chronic illness management might usefully consider the role of VCOs.

Connecting local support: A qualitative study exploring the role of voluntary organisations in long-term condition management.

OBJECTIVES: To examine the role of community groups to support people living with long-term conditions and the organisational factors that influence this role. METHODS: Thirty-three semi-structured interviews were conducted with voluntary group organisers purposefully sampled in Greater Manchester from a local database of community groups. Interviews explored the organisations role in supporting people living with a long-term condition, their social networks and the origins of the groups. RESULTS: Respondents' construed their role in supporting individual capacity for management either explicitly (e.g. providing exercise) or implicitly (e.g. emotional support). This role was influenced by a combination of group ideology, funding and social networks. Analysis highlights the role of the non-clinical setting, the social support provided within the group, as well as organisational processes that influenced their capacity to support people living with long-term conditions. CONCLUSION: By examining the organisation of voluntary groups, this study highlights the way in which they may support or constrain access to an extended range of support for people with long-term conditions. This paper has implications for commissioning of services by the health service from the third sector because of the differing ideological perspectives and limited operational capacity.

Attitudes Toward Cancer Clinical Trial Participation in Young Adults with a History of Cancer and a Healthy College Student Sample: A Preliminary Investigation.

Purpose: Adolescents and young adults (AYAs) aged 15-39 at diagnosis have very low cancer clinical trial accrual rates. To date, no studies have examined attitudes toward clinical trial participation in this age range to determine if certain individuals are less likely to enroll if offered participation. The current study assessed attitudes toward participation using the Cancer Treatment Subscale of the Attitudes toward Cancer Trials Scales. Methods: Data were collected from a sample of leukemia and lymphoma survivors (n=99) and a healthy college student sample (n=397). Following a principal components analysis, two subscales-Personal Barriers/Safety and Personal Benefits-were retained for analysis. Results: In the cancer survivor group, only 14 (13.3%) reported being offered participation in a cancer clinical trial, and only 8 of those 14 (7.6% of survivors) participated. Responses from leukemia and lymphoma survivors revealed no significant relationships between age, gender, race/ethnicity, clinical trial participation, insurance status, or social class with Personal Benefits or Personal Barriers/Safety. Healthy college females had more negative Personal Barriers/Safety attitudes compared to males after adjusting for race/ethnicity and social class (p=0.01), but no associations were present when examining Personal Benefits as an outcome. Conclusion: This preliminary investigation suggests that drivers of attitudes toward clinical trial participation in AYAs are not well understood and may impact cancer trial participation. Future work should focus on defining attitudes toward cancer clinical trials in the AYA population and developing interventions to increase awareness, knowledge, and positive attitudes toward participating in cancer research.

Multilevel socioeconomic effects on quality of life in adolescent and young adult survivors of leukemia and lymphoma.

PURPOSE: Cancer registry survival analyses have shown that adolescent and young adult patients with low socioeconomic status (SES) have reduced survival compared to those with higher SES. The objective of this study was to determine whether neighborhood- (nSES) and/or individual-level SES (iSES) also predicted current quality of life in adolescent and young adult survivors. METHODS: The Socioeconomics and Quality of Life study surveyed adolescent and young adult survivors of leukemia and lymphoma at least one year post-diagnosis using population-based ascertainment. Factor analysis was used to create a multidimensional age-relevant iSES score and compared with a preexisting census-block-group derived nSES score. Four quality of life domains were assessed: physical health, psychological and emotional well-being, social relationships, and life skills. Nested multivariable linear regression models were run to test the associations between both SES measures and quality of life and to compare the explanatory power of nSES and iSES. RESULTS: Data from 110 individuals aged 16-40 were included in the final analysis. After adjustment for sociodemographic confounders, low nSES was associated only with poorer physical health, whereas low iSES was related to poorer quality of life in all four domains with iSES accounting for an additional 14, 12, 25, and 10 % of the variance, respectively. CONCLUSIONS: Measures of SES at the individual as compared to the neighborhood level may be stronger indicators of outcomes in adolescents and young adults, which has important implications for SES measurement in the context of cancer surveillance.

A keratin 15 containing stem cell population from the hair follicle contributes to squamous papilloma development in the mouse.

The multistage model of nonmelanoma skin carcinogenesis has contributed significantly to our understanding of epithelial cancer in general. We used the Krt1-15CrePR1;R26R transgenic mouse to determine the contribution of keratin 15+ cells from the hair follicle to skin tumor development by following the labeled progeny of the keratin 15 expressing cells into papillomas. We present three novel observations. First, we found that keratin 15 expressing cells contribute to most of the papillomas by 20 weeks of promotion. Second, in contrast to the transient behavior of labeled keratin 15-derived progeny in skin wound healing, keratin 15 progeny persist in papillomas, and some malignancies for many months following transient induction of the reporter gene. Third, papillomas have surprising heterogeneity not only in their cellular composition, but also in their expression of the codon 61 signature Ha-ras mutation with approximately 30% of keratin 15-derived regions expressing the mutation. Together, these results demonstrate that keratin 15 expressing cells of the hair follicle contribute to cutaneous papillomas with long term persistence and a subset of which express the Ha-ras signature mutation characteristic of initiated cells.

CD133 in the selection of epidermal stem cells in mice: steps in the right direction.

Charruyer and colleagues (this issue) report two significant advances to the field of cutaneous keratinocyte stem cells: a pair of new selectable markers that recognize a subset of α6(+)CD34(+) label-retaining cells, and an in vivo limiting dilution assay for keratinocyte stem cells with long-term repopulating ability. This work has important implications for keratinocyte stem cell identification and assay, as well as for the identification of target cells in non-melanoma skin cancer.

Innate immunity and the regulation and mobilization of keratinocyte stem cells: are the old players playing a new game?

The skin provides an anatomical barrier to physical, chemical and biological agents. Hence, it is not surprising that it has well-developed innate immunity. What we find surprising is that the CD49f(+) /CD34(+) hair follicle stem cells should have an enriched expression profile of so many genes involved in innate immunity. Do these stem cells require extra protection from environmental insults? Or, could there be a new role for these genes? To probe these questions, we first summarize the roles of some key players in epidermal innate immunity. We next focus on their expression in CD49f(+) /CD34(+) hair follicle stem cells. Then, we consider recent data suggesting a new role for these 'old players' in the regulation and mobilization of haematopoietic and mesenchymal stem cells. Finally, we hypothesize that the 'old players' in these hair follicle stem cells may be playing a 'new game'.