Regulation of fatty acid delivery to metastases by tumor endothelium.

Tumor metastasis, the main cause of death in cancer patients, requires outgrowth of tumor cells after their dissemination and residence in microscopic niches. Nutrient sufficiency is a determinant of such outgrowth1. Fatty acids (FA) can be metabolized by cancer cells for their energetic and anabolic needs but impair the cytotoxicity of T cells in the tumor microenvironment (TME)2,3, thereby supporting metastatic progression. However, despite the important role of FA in metastatic outgrowth, the regulation of intratumoral FA is poorly understood. In this report, we show that tumor endothelium actively promotes tumor growth and restricts anti-tumor cytolysis by transferring FA into developing metastatic tumors. This process uses transendothelial fatty acid transport via endosome cargo trafficking in a mechanism that requires mTORC1 activity. Thus, tumor burden was significantly reduced upon endothelial-specific targeted deletion of Raptor, a unique component of the mTORC1 complex (RptorECKO). In vivo trafficking of a fluorescent palmitic acid analog to tumor cells and T cells was reduced in RptorECKO lung metastatic tumors, which correlated with improved markers of T cell cytotoxicity. Combination of anti-PD1 with RAD001/everolimus, at a low dose that selectively inhibits mTORC1 in endothelial cells4, impaired FA uptake in T cells and reduced metastatic disease, corresponding to improved anti-tumor immunity. These findings describe a novel mechanism of transendothelial fatty acid transfer into the TME during metastatic outgrowth and highlight a target for future development of therapeutic strategies.

STING-activating nanoparticles normalize the vascular-immune interface to potentiate cancer immunotherapy.

The tumor-associated vasculature imposes major structural and biochemical barriers to the infiltration of effector T cells and effective tumor control. Correlations between stimulator of interferon genes (STING) pathway activation and spontaneous T cell infiltration in human cancers led us to evaluate the effect of STING-activating nanoparticles (STANs), which are a polymersome-based platform for the delivery of a cyclic dinucleotide STING agonist, on the tumor vasculature and attendant effects on T cell infiltration and antitumor function. In multiple mouse tumor models, intravenous administration of STANs promoted vascular normalization, evidenced by improved vascular integrity, reduced tumor hypoxia, and increased endothelial cell expression of T cell adhesion molecules. STAN-mediated vascular reprogramming enhanced the infiltration, proliferation, and function of antitumor T cells and potentiated the response to immune checkpoint inhibitors and adoptive T cell therapy. We present STANs as a multimodal platform that activates and normalizes the tumor microenvironment to enhance T cell infiltration and function and augments responses to immunotherapy.

Loss of vascular endothelial glutaminase inhibits tumor growth and metastasis, and increases sensitivity to chemotherapy.

Glutamine is the most abundant non-essential amino acid in blood stream; yet it's concentration in tumor interstitium is markedly lower than that in the serum, reflecting the huge demand of various cell types in tumor microenvironment for glutamine. While many studies have investigated glutamine metabolism in tumor epithelium and infiltrating immune cells, the role of glutamine metabolism in tumor blood vessels remains unknown. Here, we report that inducible genetic deletion of glutaminase (GLS) specifically in host endothelium, GLSECKO, impairs tumor growth and metastatic dissemination in vivo. Loss of GLS decreased tumor microvascular density, increased perivascular support cell coverage, improved perfusion, and reduced hypoxia in mammary tumors. Importantly, chemotherapeutic drug delivery and therapeutic efficacy were improved in tumor-bearing GLSECKO hosts or in combination with GLS inhibitor, CB839. Mechanistically, loss of GLS in tumor endothelium resulted in decreased leptin levels, and exogenous recombinant leptin rescued tumor growth defects in GLSECKO mice. Together, these data demonstrate that inhibition of endothelial glutamine metabolism normalizes tumor vessels, reducing tumor growth and metastatic spread, improving perfusion, and reducing hypoxia, and enhancing chemotherapeutic delivery. Thus, targeting glutamine metabolism in host vasculature may improve clinical outcome in patients with solid tumors.

Selective glutamine metabolism inhibition in tumor cells improves antitumor T lymphocyte activity in triple-negative breast cancer.

Rapidly proliferating tumor and immune cells need metabolic programs that support energy and biomass production. The amino acid glutamine is consumed by effector T cells and glutamine-addicted triple-negative breast cancer (TNBC) cells, suggesting that a metabolic competition for glutamine may exist within the tumor microenvironment, potentially serving as a therapeutic intervention strategy. Here, we report that there is an inverse correlation between glutamine metabolic genes and markers of T cell-mediated cytotoxicity in human basal-like breast cancer (BLBC) patient data sets, with increased glutamine metabolism and decreased T cell cytotoxicity associated with poor survival. We found that tumor cell-specific loss of glutaminase (GLS), a key enzyme for glutamine metabolism, improved antitumor T cell activation in both a spontaneous mouse TNBC model and orthotopic grafts. The glutamine transporter inhibitor V-9302 selectively blocked glutamine uptake by TNBC cells but not CD8+ T cells, driving synthesis of glutathione, a major cellular antioxidant, to improve CD8+ T cell effector function. We propose a "glutamine steal" scenario, in which cancer cells deprive tumor-infiltrating lymphocytes of needed glutamine, thus impairing antitumor immune responses. Therefore, tumor-selective targeting of glutamine metabolism may be a promising therapeutic strategy in TNBC.

Microenvironmental Metabolism Regulates Antitumor Immunity.

Metabolic reprogramming of cancer cells and the tumor microenvironment are emerging as key factors governing tumor growth, metastasis, and response to therapies including immune checkpoint inhibitors. It has been recognized that rapidly proliferating cancer cells, tumor-infiltrating lymphocytes, and vascular endothelial cells compete for oxygen and nutrients. Tumor cells and other cell types in the microenvironment not only compete for nutrients, but they also simultaneously produce immunosuppressive metabolites, leading to immune escape. In addition, commensal microbial metabolites can influence regulatory T cells and inflammation in the intestine, thus playing an essential role in cancer prevention or cancer promotion. In this review, we summarize recent advances on metabolic interactions among various cell types in the tumor microenvironment, with a focus on how these interactions affect tumor immunity. We also discuss the potential role of blood vessel metabolism in regulating immune cell trafficking and activation.

Practice parameters for the diagnosis and treatment of anal intraepithelial neoplasia (AIN) on behalf of the Italian Society of Colorectal Surgery (SICCR).

Squamous cell carcinoma (SCC) of the anus is a human papilloma virus (HPV) related malignancy that is preceded by anal intraepithelial neoplasia (AIN) making this cancer, at least theoretically, a preventable disease. In the past 10 years the diagnosis, management and nomenclature of AIN has dramatically changed. Increased life expectancy in human immunodeficiency virus (HIV) positive patients due to highly active antiretroviral therapy (HAART) has caused an increase in the incidence of SCC of the anus. While many experts recommend screening and treatment of anal high-grade squamous intraepithelial lesion (HSIL), there is no consensus on the optimal management these lesions. Therefore, there is a need to review the current evidence on diagnosis and treatment of AIN and formulate recommendations to guide management. Surgeons who are members of the Italian Society of Colorectal Surgery (SICCR) with a recognized interest in AIN were invited to contribute on various topics after a comprehensive literature search. Levels of evidence were classified using the Oxford Centre for Evidence-based Medicine of 2009 and the strength of recommendation was graded according to the United States (US) preventive services task force. These recommendations are among the few entirely dedicated only to the precursors of SCC of the anus and provide an evidence-based summary of the current knowledge about the management of AIN that will serve as a reference for clinicians involved in the treatment of patients at risk for anal cancer.

The receptor tyrosine kinase EphA2 promotes glutamine metabolism in tumors by activating the transcriptional coactivators YAP and TAZ.

Malignant tumors reprogram cellular metabolism to support cancer cell proliferation and survival. Although most cancers depend on a high rate of aerobic glycolysis, many cancer cells also display addiction to glutamine. Glutamine transporters and glutaminase activity are critical for glutamine metabolism in tumor cells. We found that the receptor tyrosine kinase EphA2 activated the TEAD family transcriptional coactivators YAP and TAZ (YAP/TAZ), likely in a ligand-independent manner, to promote glutamine metabolism in cells and mouse models of HER2-positive breast cancer. Overexpression of EphA2 induced the nuclear accumulation of YAP and TAZ and increased the expression of YAP/TAZ target genes. Inhibition of the GTPase Rho or the kinase ROCK abolished EphA2-dependent YAP/TAZ nuclear localization. Silencing YAP or TAZ substantially reduced the amount of intracellular glutamate through decreased expression of SLC1A5 and GLS, respectively, genes that encode proteins that promote glutamine uptake and metabolism. The regulatory DNA elements of both SLC1A5 and GLS contain TEAD binding sites and were bound by TEAD4 in an EphA2-dependent manner. In patient breast cancer tissues, EphA2 expression positively correlated with that of YAP and TAZ, as well as that of GLS and SLC1A5 Although high expression of EphA2 predicted enhanced metastatic potential and poor patient survival, it also rendered HER2-positive breast cancer cells more sensitive to glutaminase inhibition. The findings define a previously unknown mechanism of EphA2-mediated glutaminolysis through YAP/TAZ activation in HER2-positive breast cancer and identify potential therapeutic targets in patients.

A versatile cell-penetrating peptide-adaptor system for efficient delivery of molecular cargos to subcellular destinations.

Cell penetrating peptides have long held great potential for delivery of biomolecular cargos for research, therapeutic and diagnostic purposes. They allow rapid, relatively nontoxic passage of a wide variety of biomolecules through the plasma membranes of living cells. However, CPP-based research tools and therapeutics have been stymied by poor efficiency in release from endosomes and a great deal of effort has been made to solve this 'endosomal escape problem.' Previously, we showed that use of a reversible, noncovalent coupling between CPP and cargo using calmodulin and a calmodulin binding motif allowed efficient delivery of cargo proteins to the cytoplasm in baby hamster kidney and other mammalian cell lines. The present report demonstrates the efficacy of our CPP-adaptor scheme for efficient delivery of model cargos to the cytoplasm using a variety of CPPs and adaptors. Effective overcoming of the endosomal escape problem is further demonstrated by the delivery of cargo to the nucleus, endoplasmic reticulum and peroxisomes by addition of appropriate subcellular localization signals to the cargos. CPP-adaptors were also used to deliver cargo to myotubes, demonstrating the feasibility of the system as an alternative to transfection for the manipulation of hard-to-transfect cells.

The SH3 domain of UNC-89 (obscurin) interacts with paramyosin, a coiled-coil protein, in Caenorhabditis elegans muscle.

UNC-89 is a giant polypeptide located at the sarcomeric M-line of Caenorhabditis elegans muscle. The human homologue is obscurin. To understand how UNC-89 is localized and functions, we have been identifying its binding partners. Screening a yeast two-hybrid library revealed that UNC-89 interacts with paramyosin. Paramyosin is an invertebrate-specific coiled-coil dimer protein that is homologous to the rod portion of myosin heavy chains and resides in thick filament cores. Minimally, this interaction requires UNC-89's SH3 domain and residues 294-376 of paramyosin and has a KD of ∼1.1 µM. In unc-89 loss-of-function mutants that lack the SH3 domain, paramyosin is found in accumulations. When the SH3 domain is overexpressed, paramyosin is mislocalized. SH3 domains usually interact with a proline-rich consensus sequence, but the region of paramyosin that interacts with UNC-89's SH3 is α-helical and lacks prolines. Homology modeling of UNC-89's SH3 suggests structural features that might be responsible for this interaction. The SH3-binding region of paramyosin contains a "skip residue," which is likely to locally unwind the coiled-coil and perhaps contributes to the binding specificity.

Novel cell-penetrating peptide-adaptors effect intracellular delivery and endosomal escape of protein cargos.

The use of cell-penetrating peptides (CPPs) as biomolecular delivery vehicles holds great promise for therapeutic and other applications, but development has been stymied by poor delivery and lack of endosomal escape. We have developed a CPP-adaptor system capable of efficient intracellular delivery and endosomal escape of user-defined protein cargos. The cell-penetrating sequence of HIV transactivator of transcription was fused to calmodulin, which binds with subnanomolar affinity to proteins containing a calmodulin binding site. Our strategy has tremendous advantage over prior CPP technologies because it utilizes high-affinity non-covalent, but reversible coupling between CPP and cargo. Three different cargo proteins fused to a calmodulin binding sequence were delivered to the cytoplasm of eukaryotic cells and released, demonstrating the feasibility of numerous applications in living cells including alteration of signaling pathways and gene expression.

Comparison of patients in three different rehabilitation settings after knee or hip arthroplasty: a natural observational, prospective study.

BACKGROUND: Patients after primary hip or knee replacement surgery can benefit from postoperative treatment in terms of improvement of independence in ambulation, transfers, range of motion and muscle strength. After discharge from hospital, patients are referred to different treatment destination and modalities: intensive inpatient rehabilitation (IR), cure (medically prescribed stay at a convalescence center), or ambulatory treatment (AT) at home. The purpose of this study was to 1) measure functional health (primary outcome) and function relevant factors in patients with hip or knee arthroplasty and to compare them in relation to three postoperative management strategies: AT, Cure and IR and 2) compare the post-operative changes in patient's health status (between preoperative and the 6 month follow-up) for three rehabilitation settings. METHODS: Natural observational, prospective two-center study with follow-up. Sociodemographic data and functional mobility tests, Timed Up and Go (TUG) and Iowa Level of Assistance Scale (ILOAS) of 201 patients were analysed before arthroplasty and at the end of acute hospital stay (mean duration of stay: 9.7 days +/- 3.9). Changes in health state were measured with the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) before and 6 months after arthroplasty. RESULTS: Compared to patients referred for IR and Cure, patients referred for AT were significantly younger and less comorbid. Patients admitted to IR had the highest functional disability before arthroplasty. Before rehabilitation, mean TUG was 40.0 s in the IR group, 33.9 s in the Cure group, and 27.5 s in the AT group, and corresponding mean ILOAS was 16.0, 13.0 and 12.2 (50.0 = worst). At the 6 months follow-up, the corresponding effect sizes of the WOMAC global score were 1.32, 1.87, and 1.51 (>0 means improvement). CONCLUSIONS: Age, comorbidity and functional disability are associated with referral for intensive inpatient rehabilitation after hip or knee arthroplasty and partly affect health changes after rehabilitation.

Complications during colonoscopy: prevention, diagnosis, and management.

Colonoscopy is largely performed in daily clinical practice for both diagnostic and therapeutic purposes. Although infrequent, different complications may occur during the examination, mostly related to the operative procedures. These complications range from asymptomatic and self-limiting to serious, requiring a prompt medical, endoscopic or surgical intervention. In this review, the complications that may occur during colonoscopy are discussed, with a particular focus on prevention, diagnosis, and therapeutic approaches.

Horticultural therapy for patients with chronic musculoskeletal pain: results of a pilot study.

CONTEXT: Therapists can use horticultural therapy as an adjuvant therapy in a non threatening context, with the intent of bringing about positive effects in physical health, mental health, and social interaction. Very few experimental studies exist that test its clinical effectiveness. OBJECTIVE: To determine whether the addition of horticultural therapy to a pain-management program improved physical function, mental health, and ability to cope with pain. DESIGN: The research team designed a prospective, nonrandomized, controlled cohort study, enrolling all patients consecutively referred to the Zurzach Interdisciplinary Pain Program (ZISP) who met the studys criteria. The team divided them into two cohorts based on when medical professionals referred them: before (control group) or after (intervention group) introduction of a horticultural therapy program. SETTING: The setting was the rehabilitation clinic (RehaClinic) in Bad Zurzach, Switzerland. PARTICIPANTS: Seventy-nine patients with chronic musculoskeletal pain (fibromyalgia or chronic, nonspecific back pain) participated in the study. INTERVENTIONS: The research team compared a 4-week, inpatient, interdisciplinary pain-management program with horticultural therapy (intervention, n = 37) with a pain-management program without horticultural therapy (control, n = 42). The horticultural therapy program consisted of seven sessions of group therapy, each of 1-hour duration. OUTCOME MEASURES: The research team assessed the outcome using the Medical Outcome Study Short Form-36 (SF-36), the West Haven-Yale Multidimensional Pain Inventory (MPI), the Hospital Anxiety and Depression Scale (HADS), the Coping Strategies Questionnaire (CSQ ), and two functional performance tests. The team tested participants on entry to and discharge from the 4-week pain-management program. RESULTS: Between-group differences in sociodemographic and outcome variables were not significant on participants entry to the pain-management program. On discharge, the research team measured small to moderate outcome effects (effect size [ES] up to 0.71) within both groups. The study found significantly larger improvements for the horticultural therapy group vs the control group in SF-36 role physical (ES = 0.71 vs 0.22; P = .018); SF-36 mental health (ES = 0.46 vs 0.16; P = .027); HADS anxiety (ES = 0.26 vs 0.03; P = .043); and CSQ pain behavior (ES = 0.30 vs -0.05; P = .032). CONCLUSION: The addition of horticultural therapy to a pain management program improved participants' physical and mental health and their coping ability with respect to chronic musculoskeletal pain.

Natural orifice transluminal endoscopic surgery (NOTES) and colorectal cancer?

Surgical techniques and technologies are rapidly evolving. In the field of colorectal surgery the transanal video-assisted approach was introduced by Buess, 30 years ago, with transanal endoscopic microsurgery (TEM). In more recent years different techniques and technologies have been proposed, including natural orifice specimen extraction (NOSE), natural orifice transluminal endoscopic surgery (NOTES) and single-access surgery. Furthermore, a better understanding of the prognostic and risk factors of rectal cancer has allowed TEM to expand its indications to local resection of selected tumours, and more recently there have been proposals for sentinel node biopsy in colon and rectal cancer.

Does classification of persons with fibromyalgia into Multidimensional Pain Inventory subgroups detect differences in outcome after a standard chronic pain management program?

INTRODUCTION: The present study aimed to replicate and validate the empirically derived subgroup classification based on the Multidimensional Pain Inventory (MPI) in a sample of highly disabled fibromyalgia (FM) patients. Second, it examined how the identified subgroups differed in their response to an intensive, interdisciplinary inpatient pain management program. METHODS: Participants were 118 persons with FM who experienced persistent pain and were disabled. Subgroup classification was conducted by cluster analysis using MPI subscale scores at entry to the program. At program entry and discharge, participants completed the MPI, Medical Outcomes Study Short Form-36, Hospital Anxiety and Depression Scale and Coping Strategies Questionnaire. RESULTS: Cluster analysis identified three subgroups in the highly disabled sample that were similar to those described by other studies using less disabled samples of FM. The dysfunctional subgroup (DYS; 36% of the sample) showed the highest level of depression, the interpersonally distressed subgroup (ID; 24%) showed a modest level of depression and the adaptive copers subgroup (AC; 38%) showed the lowest depression scores in the MPI (negative mood), Medical Outcomes Study Short Form-36 (mental health), Hospital Anxiety and Depression Scale (depression) and Coping Strategies Questionnaire (catastrophizing). Significant differences in treatment outcome were observed among the three subgroups in terms of reduction of pain severity (as assessed using the MPI). The effect sizes were 1.42 for DYS, 1.32 for AC and 0.62 for ID (P=0.004 for pairwise comparison of ID-AC and P=0.018 for ID-DYS). DISCUSSION: These findings underscore the importance of assessing individuals' differences in how they adjust to FM.

The effect of fluoxetine on anxiety and depression symptoms in cancer patients.

Little has been done to study the effectiveness of antidepressants in controlling anxiety/depression in a population of cancer patients. A double-blind placebo-controlled study was therefore designed to assess the effectiveness of 20 mg fluoxetine. Of 115 cancer patients who fulfilled entry criteria for levels of distress, 45 patients were randomized to a fluoxetine treatment group (FA) and 46 patients to a placebo group (PA) after a 1-week placebo period designed to exclude placebo responders. The Montgomery and Asberg Depression Scale (MADRS), the Hamilton Anxiety Scale (HAS), the Hospital Anxiety and Depression Scale (HADS), the Revised Symptom Checklist (SCL90-R) and the Spitzer Quality of Life Index (SQOLI) were used to assess the efficacy of fluoxetine. The response rate, defined by a HADS score lower than 8 after 5 weeks of treatment, was not significantly higher in the FA group (11%) compared to the PA group (7%). Compared to the PA group, patients in the FA group showed a significantly greater decrease in SCL90-R mean total score after 5 weeks, but not a greater decrease in HADS mean score. No difference between the two groups was found in observer-reported assessments (MADRS, HAS and SQOLI). Significantly more drop-outs were observed in the FA group (n = 15) than in the PA group (n = 7), although the frequencies of side-effects were not significantly different.