Host derived macrophage migration inhibitory factor expression attenuates anti-tumoral immune cell accumulation and promotes immunosuppression in the tumor microenvironment of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a significant public health concern worldwide. Immunomodulatory targets in the HNSCC tumor microenvironment are crucial to enhance the efficacy of HNSCC immunotherapy. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that has been linked to poor prognosis in many cancers, but the mechanistic role of MIF in HNSCC remains unclear. Using a murine orthotopic oral cancer model in Mif+/+ or Mif-/- mice, we determined the function of host derived MIF in HNSCC tumor development, metastasis as well as localized and systemic tumor immune responses. We observed that Mif-/- mice have decreased tumor growth and tumor burden compared to their wild-type counterparts. Flow cytometric analysis of immune populations within the primary tumor site revealed increased Th1 and cytotoxic T cell recruitment to the HNSCC tumor microenvironment. Within the tumors of Mif-/- mice, MIF deletion also enhanced the effector function of anti-tumoral effector CD8+ T cells as well as Th1 cells and decreased the accumulation of granulocytic myeloid derived suppressor cells (g-MDSCs) in the tumor microenvironment. Furthermore, MDSCs isolated from tumor bearing mice chemotactically respond to MIF in a dose dependent manner. Taken together, our results demonstrate a chemotactic and immunomodulatory role for host derived MIF in promoting HNSCC and suggest that MIF targeted immunomodulation is a promising approach for HNSCC treatment.

Ionizing Radiation Reduces Head and Neck Squamous Cell Carcinoma Cell Viability and Is Associated with Predictive Tumor-Specific T Cell Responses.

Head and neck squamous cell carcinoma (HNSCC) is common and deadly, and there is a need for improved strategies to predict treatment responses. Ionizing radiation (IR) has been demonstrated to improve HNSCC outcomes, but its effects on immune responses are not well characterized. We determined the impact of IR on T cell immune responses ex vivo. Human and mouse HNSCC cells were exposed to IR ranging from 20 to 200 Gy to determine cell viability and the ability to stimulate T-cell-specific responses. Lymph node cells of LY2 and MOC2 tumor-bearing or non-tumor-bearing mice were re-stimulated with a tumor antigen derived from LY2 or MOC2 cells treated with 200 Gy IR, ultraviolet (UV) exposure, or freeze/thaw cycle treatments. T cell proliferation and cytokine production were compared to T cells restimulated with plate-bound CD3 and CD28 antibodies. Human and mouse HNSCC cells showed reduced viability in response to ionizing radiation in a dose-dependent manner, and induced expression of T cell chemotactic cytokines. Tumor antigens derived from IR-treated LY2 and MOC2 cells induced greater proliferation of lymph node cells from tumor-bearing mice and induced unique T cell cytokine expression profiles. Our results demonstrate that IR induces potent tumoral immune responses, and IR-generated tumor antigens can potentially serve as an indicator of antitumor immune responses to HNSCC in ex vivo T cell restimulation assays.

Inducible TgfbR1 and Pten deletion in a model of tongue carcinogenesis and chemoprevention.

Head and neck squamous cell carcinoma (HNSCC) is a significant public health problem, with a need for novel approaches to chemoprevention and treatment. Preclinical models that recapitulate molecular alterations that occur in clinical HNSCC patients are needed to better understand molecular and immune mechanisms of HNSCC carcinogenesis, chemoprevention, and efficacy of treatment. We optimized a mouse model of tongue carcinogenesis with discrete quantifiable tumors via conditional deletion of Tgfßr1 and Pten by intralingual injection of tamoxifen. We characterized the localized immune tumor microenvironment, metastasis, systemic immune responses, associated with tongue tumor development. We further determined the efficacy of tongue cancer chemoprevention using dietary administration of black raspberries (BRB). Three Intralingual injections of 500 µg tamoxifen to transgenic K14 Cre, floxed Tgfbr1, Pten (2cKO) knockout mice resulted in tongue tumors with histological and molecular profiles, and lymph node metastasis similar to clinical HNSCC tumors. Bcl2, Bcl-xl, Egfr, Ki-67, and Mmp9, were significantly upregulated in tongue tumors compared to surrounding epithelial tissue. CD4+ and CD8 + T cells in tumor-draining lymph nodes and tumors displayed increased surface CTLA-4 expression, suggestive of impaired T-cell activation and enhanced regulatory T-cell activity. BRB administration resulted in reduced tumor growth, enhanced T-cell infiltration to the tongue tumor microenvironment and robust antitumoral CD8+ cytotoxic T-cell activity characterized by greater granzyme B and perforin expression. Our results demonstrate that intralingual injection of tamoxifen in Tgfßr1/Pten 2cKO mice results in discrete quantifiable tumors suitable for chemoprevention and therapy of experimental HNSCC.

Black raspberry extract inhibits regulatory T-cell activity in a murine model of head and neck squamous cell carcinoma chemoprevention.

Head and neck squamous cell carcinomas (HNSCC) are one of the most diagnosed malignancies globally, with a 5-year survival rate of approximately 40% to 50%. Current therapies are limited to highly invasive surgery, aggressive radiation, and chemotherapies. Recent reports have demonstrated the potential phytochemical properties of black raspberries in inhibiting the progression of various cancers including HNSCCs. However, the effects of black raspberry extracts on immune cells of the tumor microenvironment, specifically regulatory T cells during HNSCC, have not been investigated. We used a mouse model of 4-nitroquinoline-1-oxide (4NQO) chemically induced HNSCC carcinogenesis to determine these effects. C57BL/6 mice were exposed to 4NQO for 16 weeks and regular water for 8 weeks. 4NQO-exposed mice were fed the AIN-76A control mouse diet or the AIN76 diet supplemented with black raspberry extract. At terminal sacrifice, tumor burdens and immune cell recruitment and activity were analyzed in the tumor microenvironment, draining lymph nodes, and spleens. Mice fed the BRB extract-supplemented diet displayed decreased tumor burden compared to mice provided the AIN-76A control diet. Black raspberry extract administration did not affect overall T-cell populations as well as Th1, Th2, or Th17 differentiation in spleens and tumor draining lymph nodes. However, dietary black raspberry extract administration inhibited regulatory T-cell recruitment to HNSCC tumor sites. This was associated with an increased cytotoxic immune response in the tumor microenvironment characterized by increased CD8+ T cells and enhanced Granzyme B production during BRB extract-mediated HNSCC chemoprevention. Interestingly, this enhanced CD8+ T-cell antitumoral response was localized at the tumor sites but not at spleens and draining lymph nodes. Furthermore, we found decreased levels of PD-L1 expression by myeloid populations in draining lymph nodes of black raspberry-administered carcinogen-induced mice. Taken together, our findings demonstrate that black raspberry extract inhibits regulatory T-cell recruitment and promotes cytotoxic CD8 T-cell activity at tumor sites during HNSCC chemoprevention. These results demonstrate the immunomodulatory potential of black raspberry extracts and support the use of black raspberry-derived phytochemicals as a complementary approach to HNSCC chemoprevention and treatment.

Reduced B12 uptake and increased gastrointestinal formate are associated with archaeome-mediated breath methane emission in humans.

BACKGROUND: Methane is an end product of microbial fermentation in the human gastrointestinal tract. This gas is solely produced by an archaeal subpopulation of the human microbiome. Increased methane production has been associated with abdominal pain, bloating, constipation, IBD, CRC or other conditions. Twenty percent of the (healthy) Western populations innately exhale substantially higher amounts (>5 ppm) of this gas. The underlying principle for differential methane emission and its effect on human health is not sufficiently understood. RESULTS: We assessed the breath methane content, the gastrointestinal microbiome, its function and metabolome, and dietary intake of one-hundred healthy young adults (female: n = 52, male: n = 48; mean age =24.1). On the basis of the amount of methane emitted, participants were grouped into high methane emitters (CH4 breath content 5-75 ppm) and low emitters (CH4 < 5 ppm). The microbiomes of high methane emitters were characterized by a 1000-fold increase in Methanobrevibacter smithii. This archaeon co-occurred with a bacterial community specialized on dietary fibre degradation, which included members of Ruminococcaceae and Christensenellaceae. As confirmed by metagenomics and metabolomics, the biology of high methane producers was further characterized by increased formate and acetate levels in the gut. These metabolites were strongly correlated with dietary habits, such as vitamin, fat and fibre intake, and microbiome function, altogether driving archaeal methanogenesis. CONCLUSIONS: This study enlightens the complex, multi-level interplay of host diet, genetics and microbiome composition/function leading to two fundamentally different gastrointestinal phenotypes and identifies novel points of therapeutic action in methane-associated disorders. Video Abstract.

Metabolic profiling links cardiovascular risk and vascular end organ damage.

BACKGROUND AND AIMS: An untargeted metabolomics approach allows for a better understanding and identification of new candidate metabolites involved in the etiology of vascular disease. We aimed to investigate the associations of cardiovascular (CV) risk factors with the metabolic fingerprint and macro- and microvascular health in an untargeted metabolomic approach in predefined CV risk groups of aged individuals. METHODS: The metabolic fingerprint and the macro- and microvascular health from 155 well-characterized aged (50-80 years) individuals, based on the EXAMIN AGE study, were analysed. Nuclear magnetic resonance spectroscopy was used to analyse the metabolic fingerprint. Carotid-femoral pulse wave velocity and retinal vessel diameters were assessed to quantify macro- and microvascular health. RESULTS: The metabolic fingerprint became more heterogeneous with an increasing number of risk factors. There was strong evidence for higher levels of glutamine [estimate (95% CI): -14.54 (-17.81 to -11.27), p < 0.001], glycine [-5.84 (-7.88 to -3.79), p < 0.001], histidine [-0.73 (-0.96 to -0.50), p < 0.001], and acetate [-1.68 (-2.91 to -0.46), p = 0.007] to be associated with a lower CV risk profile. Tryptophan, however, was positively associated with higher CV risk [0.31 (0.06-0.56), p = 0.015]. The combination of a priori defined CV risk factors explained up to 45.4% of the metabolic variation. The metabolic fingerprint explained 20% of macro- and 23% of microvascular variation. CONCLUSIONS: Metabolic profiling has the potential to improve CV risk stratification by identifying new underlying metabolic pathways associated with atherosclerotic disease development, from cardiovascular risk to metabolites, to vascular end organ damage.

Erratum: Potassium ions promote hexokinase-II dependent glycolysis.

[This corrects the article DOI: 10.1016/j.isci.2021.102346.].

Potassium ions promote hexokinase-II dependent glycolysis.

High expression levels of mitochondria-associated hexokinase-II (HKII) represent a hallmark of metabolically highly active cells such as fast proliferating cancer cells. Typically, the enzyme provides a crucial metabolic switch towards aerobic glycolysis. By imaging metabolic activities on the single-cell level with genetically encoded fluorescent biosensors, we here demonstrate that HKII activity requires intracellular K+. The K+ dependency of glycolysis in cells expressing HKII was confirmed in cell populations using extracellular flux analysis and nuclear magnetic resonance-based metabolomics. Reductions of intracellular K+ by gramicidin acutely disrupted HKII-dependent glycolysis and triggered energy stress pathways, while K+ re-addition promptly restored glycolysis-dependent adenosine-5'-triphosphate generation. Moreover, expression and activation of KV1.3, a voltage-gated K+ channel, lowered cellular K+ content and the glycolytic activity of HEK293 cells. Our findings unveil K+ as an essential cofactor of HKII and provide a mechanistic link between activities of distinct K+ channels and cell metabolism.

Endothelial Lipase Modulates Paraoxonase 1 Content and Arylesterase Activity of HDL.

Endothelial lipase (EL) is a strong modulator of the high-density lipoprotein (HDL) structure, composition, and function. Here, we examined the impact of EL on HDL paraoxonase 1 (PON1) content and arylesterase (AE) activity in vitro and in vivo. The incubation of HDL with EL-overexpressing HepG2 cells decreased HDL size, PON1 content, and AE activity. The EL modification of HDL did not diminish the capacity of HDL to associate with PON1 when EL-modified HDL was incubated with PON1-overexpressing cells. The overexpression of EL in mice significantly decreased HDL serum levels but unexpectedly increased HDL PON1 content and HDL AE activity. Enzymatically inactive EL had no effect on the PON1 content of HDL in mice. In healthy subjects, EL serum levels were not significantly correlated with HDL levels. However, HDL PON1 content was positively associated with EL serum levels. The EL-induced changes in the HDL-lipid composition were not linked to the HDL PON1 content. We conclude that primarily, the interaction of enzymatically active EL with HDL, rather than EL-induced alterations in HDL size and composition, causes PON1 displacement from HDL in vitro. In vivo, the EL-mediated reduction of HDL serum levels and the consequently increased PON1-to-HDL ratio in serum increase HDL PON1 content and AE activity in mice. In humans, additional mechanisms appear to underlie the association of EL serum levels and HDL PON1 content.

Alternate Day Fasting Improves Physiological and Molecular Markers of Aging in Healthy, Non-obese Humans.

Caloric restriction and intermittent fasting are known to prolong life- and healthspan in model organisms, while their effects on humans are less well studied. In a randomized controlled trial study (ClinicalTrials.gov identifier: NCT02673515), we show that 4 weeks of strict alternate day fasting (ADF) improved markers of general health in healthy, middle-aged humans while causing a 37% calorie reduction on average. No adverse effects occurred even after >6 months. ADF improved cardiovascular markers, reduced fat mass (particularly the trunk fat), improving the fat-to-lean ratio, and increased ß-hydroxybutyrate, even on non-fasting days. On fasting days, the pro-aging amino-acid methionine, among others, was periodically depleted, while polyunsaturated fatty acids were elevated. We found reduced levels sICAM-1 (an age-associated inflammatory marker), low-density lipoprotein, and the metabolic regulator triiodothyronine after long-term ADF. These results shed light on the physiological impact of ADF and supports its safety. ADF could eventually become a clinically relevant intervention.

Physician gender differences in general and cancer-specific prevention attitudes and practices.

BACKGROUND: Findings are inconsistent regarding physician gender differences in general prevention practices and cancer-specific attitudes and practices. METHODS: We analyzed cross-sectional data from randomly selected physicians (N = 722) to test associations of gender with prevention practices and attitudes. RESULTS: Chi-square analyses (P < .05) showed gender differences for 14% (7/49) of the general and cancer-specific practices and attitudes tested. Multivariate analyses revealed that gender significantly (P < .05) predicted general prevention practices and cancer-specific attitudes in 4 models. Female gender predicted discussion of physical activity, violence, and use of substances. Male gender predicted belief in effectiveness of prostate-specific antigen screening. CONCLUSIONS: Overall, male and female physicians showed more similarities than differences, but physician gender was associated with a number of important general and cancer-specific prevention services. Female physicians were more likely to discuss general health prevention activities than male physicians, especially issues considered sensitive. We discuss implications for research and education.

Developing a culturally competent peer support intervention for Spanish-speaking Latinas with breast cancer.

Little research exists on the need for, barriers to, and acceptability and effectiveness of psychosocial support services among Latinas with breast cancer, despite their increased risks of psychosocial distress. This formative research study identifies barriers to and benefits and components of an effective peer support counselor intervention for Spanish-speaking Latinas recently diagnosed with breast cancer. Analysis was based on interviews of 89 Latino cancer patients referred to psychosocial services; 29 Spanish-speaking survivors of breast cancer; and 17 culturally competent advocates for Latinos with cancer. Results indicate that interventions should begin close to diagnosis; build self-care skills; be culturally competent and emotionally supportive; provide language appropriate cancer information; encourage self-expression; and address lack of access to and knowledge of services. Creating such psychosocial programs with input from survivors and advocates who have similar self-identities to patients would improve quality of life in diverse and underserved populations.

Identification of limited English proficient patients in clinical care.

BACKGROUND: Standardized means to identify patients likely to benefit from language assistance are needed. OBJECTIVE: To evaluate the accuracy of the U.S. Census English proficiency question (Census-LEP) in predicting patients' ability to communicate effectively in English. DESIGN: We investigated the sensitivity and specificity of the Census-LEP alone or in combination with a question on preferred language for medical care for predicting patient-reported ability to discuss symptoms and understand physician recommendations in English. PARTICIPANTS: Three hundred and two patients > 18 who spoke Spanish and/or English recruited from a cardiology clinic and an inpatient general medical-surgical ward in 2004-2005. RESULTS: One hundred ninety-eight (66%) participants reported speaking English less than "very well" and 166 (55%) less than "well"; 157 (52%) preferred receiving their medical care in Spanish. Overall, 135 (45%) were able to discuss symptoms and 143 (48%) to understand physician recommendations in English. The Census-LEP with a high-threshold (less than "very well") had the highest sensitivity for predicting effective communication (100% Discuss; 98.7% Understand), but the lowest specificity (72.6% Discuss; 67.1% Understand). The composite measure of Census-LEP and preferred language for medical care provided a significant increase in specificity (91.9% Discuss; 83.9% Understand), with only a marginal decrease in sensitivity (99.4% Discuss; 96.7% Understand). CONCLUSIONS: Using the Census-LEP item with a high-threshold of less than "very well" as a screening question, followed by a language preference for medical care question, is recommended for inclusive and accurate identification of patients likely to benefit from language assistance.

Clinical trials attitudes and practices of Latino physicians.

BACKGROUND: Ethnic differences in physicians' attitudes and behaviors related to clinical trials might partially account for disparities in clinical trial participation among Latino patients. Literature regarding Latino physicians' clinical trials attitudes and practices, in comparison to White physicians, was lacking. METHODS: Cross-sectional data from randomly selected physicians (N=695), stratified by ethnicity, were analyzed to test associations of ethnicity with physicians' participation in and attitudes toward referral of patients to clinical trials. RESULTS: Chi-square analyses showed significant (p<0.05) associations of physician race/ethnicity and clinical trials involvement, type of trial for which the physician is likely to recommend a patient, belief in scientific value, and factors that would influence recommendation for a patient to participate. Multivariate analyses resulted in several significant (p<0.05) predictors of clinical trials outcomes, including physician race/ethnicity. CONCLUSIONS: Latino physicians were significantly less involved in clinical trials than White physicians and found less scientific value in them, highlighting areas for future education and intervention.

Information exchange and decision making in the treatment of Latina and white women with ductal carcinoma in situ.

The natural history of ductal carcinoma in situ (DCIS) is largely unknown, and its optimal treatment remains controversial. Using semi-structured interviews, this study compared 18 White and 16 Latina women's understanding of their DCIS diagnosis, treatment decision-making processes, and satisfaction with care. Ethnic differences were observed in cognitive and emotional responses to DCIS, with White women generally reporting a better understanding of their diagnosis and treatment, and Latinas reporting more distress. Regardless of ethnicity, women with DCIS preferred that physicians discuss treatment options and attend to their informational and emotional needs. Satisfaction was associated with adequate information, expediency of care, and physicians' sensitivity to patients' emotional needs.

Understanding Latino adolescent risk behaviors: parental and peer influences.

OBJECTIVE: To assess baseline factors associated with having ever drunk alcohol, smoked, and having had sex two years later among a sample of Latino adolescents. DESIGN: In a prospective cohort study, Latino adolescents completed telephone surveys assessing demographic information and health-enhancing and -compromising behaviors, administered 3 times (baseline, T2, and T3) during a two-year period. SETTING: Students were recruited between 1997 and 1998, from four middle schools within three Los Angeles school districts. PARTICIPANTS: Latino adolescents in 7th and 8th grade, from any of the four middle schools, whose parents provided written permission for them to participate in a telephone health behavior survey. MAIN OUTCOME MEASURES: Self-report of ever drank alcohol, ever smoked cigarettes, ever had sex by T3. RESULTS: Being more acculturated, engaging in risky behaviors, valuing independence, and having friends who had ever smoked at baseline were positively associated with having ever drunk alcohol and having ever smoked by T3. Parents' negative reactions to risky and unhealthy behaviors were protective against drinking and smoking. Working at a paid job and having parents with a high school education or higher were associated with drinking alcohol by T3. Older age at baseline was positively associated with having sex, while receiving good grades and valuing religion were protective against having sex. CONCLUSIONS: Findings reveal that both parents and peers are important influences on adolescent risk behaviors and suggest that interventions for adolescents to prevent such behaviors should involve peers and parents.

Interpersonal processes of care survey: patient-reported measures for diverse groups.

OBJECTIVE: To create a patient-reported, multidimensional physician/patient interpersonal processes of care (IPC) instrument appropriate for patients from diverse racial/ethnic groups that allows reliable, valid, and unbiased comparisons across these groups. DATA SOURCE/DATA COLLECTION: Data were collected by telephone interview. The survey was administered in English and Spanish to adult general medicine patients, stratified by race/ethnicity and language (African Americans, English-speaking Latinos, Spanish-speaking Latinos, non-Latino whites) (N=1,664). STUDY DESIGN/METHODS: In this cross-sectional study, items were designed to be appropriate for diverse ethnic groups based on focus groups, our prior framework, literature, and cognitive interviews. Multitrait scaling and confirmatory factor analysis were used to examine measurement invariance; we identified scales that allowed meaningful quantitative comparisons across four race/ethnic/language groups. PRINCIPAL FINDINGS: The final instrument assesses several subdomains of communication, patient-centered decision making, and interpersonal style. It includes 29 items representing 12 first-order and seven second-order factors with equivalent meaning (metric invariance) across groups; 18 items (seven factors) allowed unbiased mean comparison across groups (scalar invariance). Final scales exhibited moderate to high reliability. CONCLUSIONS: The IPC survey can be used to describe disparities in interpersonal care, predict patient outcomes, and examine outcomes of quality improvement efforts to reduce health care disparities.

Missed opportunities: family history and behavioral risk factors in breast cancer risk assessment among a multiethnic group of women.

BACKGROUND: Clinician's knowledge of a woman's cancer family history (CFH) and counseling about health-related behaviors (HRB) is necessary for appropriate breast cancer care. OBJECTIVE: To evaluate whether clinicians solicit CFH and counsel women on HRB; to assess relationship of well visits and patient risk perception or worry with clinician's behavior. DESIGN: Cross-sectional population-based telephone survey. PARTICIPANTS: Multiethnic sample; 1,700 women from San Francisco Mammography Registry with a screening mammogram in 2001-2002. PREDICTORS: well visit in prior year, self-perception of 10-year breast cancer risk, worry scale. OUTCOMES: Patient report of clinician asking about CFH in prior year, or ever counseling about HRB in relation to breast cancer risk. Multivariate models included age, ethnicity, education, language of interview, insurance/mammography facility, well visit, ever having a breast biopsy/follow-up mammography, Gail-Model risk, Jewish heritage, and body mass index. RESULTS: 58% reported clinicians asked about CFH; 33% reported clinicians ever discussed HRB. In multivariate analysis, regardless of actual risk, perceived risk, or level of worry, having had a well visit in prior year was associated with increased odds (OR = 2.3; 95% CI 1.6, 3.3) that a clinician asked about CFH. Regardless of actual risk of breast cancer, a higher level of worry (OR = 1.9; 95% CI 1.4, 2.6) was associated with increased odds that a clinician ever discussed HRB. CONCLUSIONS: Clinicians are missing opportunities to elicit family cancer histories and counsel about health-related behaviors and breast cancer risk. Preventive health visits offer opportunities for clinicians to address family history, risk behaviors, and patients' worries about breast cancer.

Use of cancer support groups among Latina breast cancer survivors.

INTRODUCTION: Although Latina women diagnosed with breast cancer may be at greater risk of psychosocial morbidity compared to white women, few utilize support services such as support groups. Reasons for this under-use among Latinas are unknown. METHODS: A cross-sectional telephone survey examined the association of predisposing, enabling, and need factors with use of cancer support groups among 330 Latina breast cancer survivors recruited from a population-based tumor registry in counties with Spanish language support groups. RESULTS: Thirty-two percent had ever used a support group. Among the 225 (68%) women who had never used one, major reasons for not using a support group included receiving enough support from other sources (20%), not needing one (18%), and being unaware of groups in their local area (17%). Women receiving quite a bit or a lot of encouragement from family members to attend a cancer support group were seven times more likely to have ever attended one than women receiving little or no encouragement (OR = 7.04, 95% CI 3.72, 13.30). Spiritual well-being was inversely associated with ever having attended a support group (OR = 0.93, 95% CI 0.89, 0.98). DISCUSSION: Results suggest that families play an important role in promoting use of support groups among Latina breast cancer survivors, and that spirituality may offer an alternative source of support. IMPLICATIONS FOR CANCER SURVIVORS: More effort should be directed toward providing culturally and linguistically appropriate support services to breast cancer survivors, and increasing awareness of these services among oncologists, patients and family members.