Deep Phenotyping the Anterior Urethral Stricture: Characterizing the Relationship Between Inflammation, Fibrosis, Patient History, and Disease Pathophysiology.

PURPOSE: Anterior urethral stricture disease (aUSD) is a complex, heterogeneous condition that is idiopathic in origin for most men. This gap in knowledge rarely affects the current management strategy for aUSD, as urethroplasty does not generally consider etiology. However, as we transition towards personalized, minimally invasive treatments for aUSD and begin to consider aUSD prevention strategies, disease pathophysiology will become increasingly important. The purpose of this study was to perform a deep phenotype of men undergoing anterior urethroplasty for aUSD. We hypothesized that unique biologic signatures and potential targets for intervention would emerge based on stricture presence/absence, stricture etiology, and the presence/absence of stricture inflammation. MATERIALS AND METHODS: Men with aUSD undergoing urethroplasty were recruited from one of 5 participating centers. Enrollees provided urethral stricture tissue and blood/serum on the day of surgery and completed patient-reported outcome measure questionnaires both pre- and postoperatively. The initial study had 3 aims: (1) to determine pediatric and adult subacute and repeated perineal trauma (SRPT) exposures using a study-specific SRPT questionnaire, (2) to determine the degree of inflammation and fibrosis in aUSD and peri-aUSD (normal urethra) tissue, and (3) to determine levels of systemic inflammatory and fibrotic cytokines. Two controls groups provided serum (normal vasectomy patients) and urethral tissue (autopsy patients). Cohorts were based on the presence/absence of stricture, by presumed stricture etiology (idiopathic, traumatic/iatrogenic, lichen sclerosus [LS]), and by the presence/absence of stricture inflammation. RESULTS: Of 138 enrolled men (120 tissue/serum; 18 stricture tissue only), 78 had idiopathic strictures, 33 had trauma-related strictures, and 27 had LS-related strictures. BMI, stricture length, and stricture location significantly differed between cohorts (P < .001 for each). The highest BMIs and the longest strictures were observed in the LS cohort. SRPT exposures did not significantly differ between etiology cohorts, with > 60% of each reporting low/mild risk. Stricture inflammation significantly differed between cohorts, with mild to severe inflammation present in 27% of trauma-related strictures, 54% of idiopathic strictures, and 48% of LS strictures (P = .036). Stricture fibrosis did not significantly differ between cohorts (P = .7). Three serum cytokines were significantly higher in patients with strictures compared to stricture-free controls: interleukin-9 (IL-9; P = .001), platelet-derived growth factor-BB (P = .004), and CCL5 (P = .01). No differences were observed in the levels of these cytokines based on stricture etiology. However, IL-9 levels were significantly higher in patients with inflamed strictures than in patients with strictures lacking inflammation (P = .019). Degree of stricture inflammation positively correlated with serum levels of IL-9 (Spearman's rho 0.224, P = .014). CONCLUSIONS: The most common aUSD etiology is idiopathic. Though convention has implicated SRPT as causative for idiopathic strictures, here we found that patients with idiopathic strictures had low SRPT rates that were similar to rates in patients with a known stricture etiology. Stricture and stricture-adjacent inflammation in idiopathic stricture were similar to LS strictures, suggesting shared pathophysiologic mechanisms. IL-9, platelet-derived growth factor-BB, and CCL5, which were elevated in patients with strictures, have been implicated in fibrotic conditions elsewhere in the body. Further work will be required to determine if this shared biologic signature represents a potential mechanism for an aUSD predisposition.

Loss of Nf1 and Ink4a/Arf Are Associated with Sex-Dependent Growth Differences in a Mouse Model of Embryonal Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is an aggressive form of cancer that accounts for half of all pediatric soft tissue sarcomas. Little progress has been made in improving survival outcomes over the past three decades. Mouse models of rhabdomyosarcoma are a critical component of translational research aimed at understanding tumor biology and developing new, improved therapies. Though several models exist, many common mutations found in human rhabdomyosarcoma tumors remain unmodeled and understudied. This study describes a new model of embryonal rhabdomyosarcoma driven by the loss of Nf1 and Ink4a/Arf, two mutations commonly found in patient tumors. We find that this new model is histologically similar to other previously-published rhabdomyosarcoma models, although it substantially differs in the time required for tumor onset and in tumor growth kinetics. We also observe unique sex-dependent phenotypes in both primary and newly-developed orthotopic syngeneic allograft tumors that are not present in previous models. Using in vitro and in vivo studies, we examined the response to vincristine, a component of the standard-of-care chemotherapy for RMS. The findings from this study provide valuable insight into a new mouse model of rhabdomyosarcoma that addresses an ongoing need for patient-relevant animal models to further translational research.

Variability in Methodology of Erectile Dysfunction Regenerative Therapy Trials on ClinicalTrials.gov.

OBJECTIVE: To evaluate the variability in the criteria of erectile dysfunction (ED) regenerative therapy trials registered on ClinicalTrials.gov. METHODS: Interventional trials on ClinicalTrials.gov with the keywords "erectile dysfunction" and variations of "shockwave," "platelet rich plasma," "stem cell," "regenerative," and "restorative" were examined. Inclusion/exclusion criteria and primary/secondary outcomes were compared between extracorporeal shockwave therapy (ESWT), platelet rich plasma and stem cell injections (PRP/SC), and other regenerative therapies (ORT) groups. RESULTS: Of the 92 trials analyzed, International Index of Erectile Function (IIEF) score was the most common primary outcome (72%), with a higher prevalence in ESWT trials than PRP/SC or ORT trials (89% vs 44% and 58%, P <.001). Safety/tolerability was a primary outcome for 44% of PRP/SC trials and 25% of ORT trials but no ESWT trials (P <.001). ESWT trials more frequently had sexual/romantic relationship-based inclusion criteria and cancer treatment-related exclusion criteria than PRP/SC and ORT trials. CONCLUSION: There is substantial variability in the inclusion/exclusion criteria and outcome measures among ED regenerative therapy trials. ESWT trials most frequently utilized IIEF and had the strictest inclusion/exclusion criteria, suggesting more rigorous and functional outcome-based studies. Conversely, PRP/SC and ORT trials, but not ESWT trials, had safety/tolerability as a primary outcome, likely due to the experimental nature of these therapies. The variability in inclusion/exclusion criteria and outcome measures limits comparison of the various ED regenerative therapies.

Novel Men's Health Curriculum Increases Fraternity Members' Knowledge, Confidence, and Willingness to Seek Help for Health Concerns.

OBJECTIVE: To evaluate fraternity members' men's health knowledge, confidence in understanding, and the likelihood of seeking help for men's health concerns and to assess the impact of a novel men's health curriculum on each of these measures. METHODS: Members of 6 undergraduate fraternities (n = 189) viewed a 45-minute presentation about men's health topics and completed before and after surveys. RESULTS: The presentation increased men's health knowledge, confidence in understanding men's health concerns and when/where to seek help, and the likelihood of seeking help for men's health concerns. Health knowledge did not correlate with confidence or likelihood of seeking help. Confidence positively correlated with the likelihood of seeking help before and after the presentation. CONCLUSION: A short presentation on common men's health topics increases health knowledge, confidence, and the likelihood of seeking help for these concerns. Increased confidence in understanding, rather than health knowledge, was associated with an increased willingness to seek help.

Discrepancies in indel software resolution with somatic CRISPR/Cas9 tumorigenesis models.

CRISPR/Cas9 gene editing has evolved from a simple laboratory tool to a powerful method of in vivo genomic engineering. As the applications of CRISPR/Cas9 technology have grown, the need to characterize the breadth and depth of indels generated by editing has expanded. Traditionally, investigators use one of several publicly-available platforms to determine CRISPR/Cas9-induced indels in an edited sample. However, to our knowledge, there has not been a cross-platform comparison of available indel analysis software in samples generated from somatic in vivo mouse models. Our group has pioneered using CRISPR/Cas9 to generate somatic primary mouse models of malignant peripheral nerve sheath tumors (MPNSTs) through genetic editing of Nf1. Here, we used sequencing data from the in vivo editing of the Nf1 gene in our CRISPR/Cas9 tumorigenesis model to directly compare results across four different software platforms. By analyzing the same genetic target across a wide panel of cell lines with the same sequence file, we are able to draw systematic conclusions about the differences in these software programs for analysis of in vivo-generated indels. Surprisingly, we report high variability in the reported number, size, and frequency of indels across each software platform. These data highlight the importance of selecting indel analysis platforms specific to the context that the gene editing approach is being applied. Taken together, this analysis shows that different software platforms can report widely divergent indel data from the same sample, particularly if larger indels are present, which are common in somatic, in vivo CRISPR/Cas9 tumor models.

Low-Cost, High-Pressure-Synthesized Oxygen-Entrapping Materials to Improve Treatment of Solid Tumors.

Tumor hypoxia drives resistance to many cancer therapies, including radiotherapy and chemotherapy. Methods that increase tumor oxygen pressures, such as hyperbaric oxygen therapy and microbubble infusion, are utilized to improve the responses to current standard-of-care therapies. However, key obstacles remain, in particular delivery of oxygen at the appropriate dose and with optimal pharmacokinetics. Toward overcoming these hurdles, gas-entrapping materials (GeMs) that are capable of tunable oxygen release are formulated. It is shown that injection or implantation of these materials into tumors can mitigate tumor hypoxia by delivering oxygen locally and that these GeMs enhance responsiveness to radiation and chemotherapy in multiple tumor types. This paper also demonstrates, by comparing an oxygen (O2 )-GeM to a sham GeM, that the former generates an antitumorigenic and immunogenic tumor microenvironment in malignant peripheral nerve sheath tumors. Collectively the results indicate that the use of O2 -GeMs is promising as an adjunctive strategy for the treatment of solid tumors.

PRC2 loss drives MPNST metastasis and matrix remodeling.

The histone methyltransferase PRC2 plays a complex role in cancer. Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas with frequent loss-of-function mutations in PRC2 that are associated with poor outcome. Here, we identify a critical role for PRC2 loss in driving MPNST metastasis. PRC2-dependent metastatic phenotypes included increased collagen-dependent invasion, upregulation of matrix-remodeling enzymes, and elevated lung metastasis in orthotopic mouse models. Furthermore, clinical sample analysis determined that PRC2 loss correlated with metastatic disease, increased fibrosis, and decreased survival in patients with MPNSTs. These results may have broad implications for PRC2 function across multiple cancers and provide a strong rationale for investigating potential therapies targeting ECM-remodeling enzymes and tumor fibrosis to improve outcomes in patients with MPNSTs.

Augmenting chemotherapy with low-dose decitabine through an immune-independent mechanism.

The DNA methyltransferase inhibitor decitabine has classically been used to reactivate silenced genes and as a pretreatment for anticancer therapies. In a variation of this idea, this study explores the concept of adding low-dose decitabine (DAC) following administration of chemotherapy to bolster therapeutic efficacy. We find that addition of DAC following treatment with the chemotherapy agent gemcitabine improves survival and slows tumor growth in a mouse model of high-grade sarcoma. Unlike prior studies in epithelial tumor models, DAC did not induce a robust antitumor T cell response in sarcoma. Furthermore, DAC synergizes with gemcitabine independently of the immune system. Mechanistic analyses demonstrate that the combination therapy induces biphasic cell cycle arrest and apoptosis. Therapeutic efficacy was sequence dependent, with gemcitabine priming cells for treatment with DAC through inhibition of ribonucleotide reductase. This study identifies an apparently unique application of DAC to augment the cytotoxic effects of conventional chemotherapy in an immune-independent manner. The concepts explored in this study represent a promising paradigm for cancer treatment by augmenting chemotherapy through addition of DAC to increase tolerability and improve patient response. These findings have widespread implications for the treatment of sarcomas and other aggressive malignancies.

Divergent immune landscapes of primary and syngeneic Kras-driven mouse tumor models.

Immune cells play critical functions in cancer, and mice with intact immune systems are vital to understanding tumor immunology. Both genetically engineered mouse models (GEMMs) and syngeneic cell transplant approaches use immunocompetent mice to define immune-dependent events in tumor development and progression. Due to their rapid and reproducible nature, there is expanded interest in developing new syngeneic tools from established primary tumor models. However, few studies have examined the extent that syngeneic tumors reflect the immune profile of their originating primary models. Here, we describe comprehensive immunophenotyping of two well-established GEMMs and four new syngeneic models derived from these parental primary tumors. To our knowledge, this is the first systematic analysis comparing immune landscapes between primary and orthotopic syngeneic tumors. These models all use the same well-defined human-relevant driver mutations, arise at identical orthotopic locations, and are generated in mice of the same background strain. This allows for a direct and focused comparison of tumor immune landscapes in carefully controlled mouse models. We identify key differences between the immune infiltrate of GEMM models and their corresponding syngeneic tumors. Most notable is the divergence of T cell populations, with different proportions of CD8+ T cells and regulatory T cells across several models. We also observe immune variation across syngeneic tumors derived from the same primary model. These findings highlight the importance of immune variance across mouse modeling approaches, which has strong implications for the design of rigorous and reproducible translational studies.

Vascular pedicled flaps for skull base defect reconstruction.

OBJECTIVE: Techniques for reconstruction of skull base defects have advanced greatly since the introduction of the vascular pedicled nasoseptal flap in 2006. The objective of this review is to assess the current state of the field by examining both intranasal and extranasal techniques of vascular pedicled skull base defect repair, their indications and success rates, and novel techniques that are currently under investigation. METHODS: A review of the literature describing the use of vascular pedicled flaps in skull base defect reconstruction was conducted using PubMed and Google Scholar. RESULTS: The nasoseptal flap remains the most widely used vascular pedicled flap for endoscopic repair of skull base defects. Its ease of harvest, wide arch of rotation, and high success rates make it a popular choice among surgeons. Several variations including a "rescue" nasopseptal flap have been developed. Other less commonly used pedicled intranasal flaps include the middle turbinate flap and the posterior pedicled inferior turbinate flap. Additionally, several novel vascular pedicled flaps have been developed and tested in small cohorts of patients. Extranasal flaps such as the pericranial flap and the temporoparietal fascia flap are used less frequently than intranasal flaps. However, they remain valuable options for reconstruction in certain situations. CONCLUSION: Advancements continue to be made in the field of skull base defect reconstruction using vascular pedicled flaps. Though the nasoseptal flap remains the most widely utilized option, additional intranasal techniques continue to be developed and tested to optimize surgical outcomes and patient care. LEVEL OF EVIDENCE: NA.

Distinct Tumor Microenvironments Are a Defining Feature of Strain-Specific CRISPR/Cas9-Induced MPNSTs.

The tumor microenvironment plays important roles in cancer biology, but genetic backgrounds of mouse models can complicate interpretation of tumor phenotypes. A deeper understanding of strain-dependent influences on the tumor microenvironment of genetically-identical tumors is critical to exploring genotype-phenotype relationships, but these interactions can be difficult to identify using traditional Cre/loxP approaches. Here, we use somatic CRISPR/Cas9 tumorigenesis approaches to determine the impact of mouse background on the biology of genetically-identical malignant peripheral nerve sheath tumors (MPNSTs) in four commonly-used inbred strains. To our knowledge, this is the first study to systematically evaluate the impact of host strain on CRISPR/Cas9-generated mouse models. Our data identify multiple strain-dependent phenotypes, including changes in tumor onset and the immune microenvironment. While BALB/c mice develop MPNSTs earlier than other strains, similar tumor onset is observed in C57BL/6, 129X1 and 129/SvJae mice. Indel pattern analysis demonstrates that indel frequency, type and size are similar across all genetic backgrounds. Gene expression and IHC analysis identify multiple strain-dependent differences in CD4+ T cell infiltration and myeloid cell populations, including M2 macrophages and mast cells. These data highlight important strain-specific phenotypes of genomically-matched MPNSTs that have implications for the design of future studies using similar in vivo gene editing approaches.

Locally invasive, castrate-resistant prostate cancer in a Pten/Trp53 double knockout mouse model of prostate cancer monitored with non-invasive bioluminescent imaging.

Here we have improved an existing mouse model of prostate cancer based on prostate-specific deletion of Pten and Trp53 by incorporating a Cre-activatable luciferase reporter. By coupling the deletion of those genes to the activation of a luciferase reporter, we were able to monitor tumor burden non-invasively over time. We show that, consistent with previous reports, deletion of both Pten and Trp53 on a C57BL/6 background accelerates tumor growth and results in both the loss of androgen receptor expression and castrate resistant tumors as compared with loss of Pten alone. Loss of Trp53 results in the development of sarcomatoid histology and the expression of markers of epithelial-to-mesenchymal transition Zeb1 and vimentin, with kinetics and penetrance dependent on whether one or both alleles of Trp53 were deleted. Homozygous deletion of Trp53 and Pten resulted in uniformly lethal disease by 25 weeks. While we were able to detect locally invasive disease in the peritoneal cavity in aggressive tumors from the double knockout mice, we were unable to detect lymphatic or hematogenous metastatic disease in lymph nodes or at distant sites.

Tumor Subtype Determines Therapeutic Response to Chimeric Polypeptide Nanoparticle-based Chemotherapy in Pten-deleted Mouse Models of Sarcoma.

PURPOSE: Nanoparticle-encapsulated drug formulations can improve responses to conventional chemotherapy by increasing drug retention within the tumor and by promoting a more effective antitumor immune response than free drug. New drug delivery modalities are needed in sarcomas because they are often chemoresistant cancers, but the rarity of sarcomas and the complexity of diverse subtypes makes it challenging to investigate novel drug formulations. EXPERIMENTAL DESIGN: New drug formulations can be tested in animal models of sarcomas where the therapeutic response of different formulations can be compared using mice with identical tumor-initiating mutations. Here, using Cre/loxP and CRISPR/Cas9 techniques, we generated two distinct mouse models of Pten-deleted soft-tissue sarcoma: malignant peripheral nerve sheath tumor (MPNST) and undifferentiated pleomorphic sarcoma (UPS). We used these models to test the efficacy of chimeric polypeptide doxorubicin (CP-Dox), a nanoscale micelle formulation, in comparison with free doxorubicin. RESULTS: The CP-Dox formulation was superior to free doxorubicin in MPNST models. However, in UPS tumors, CP-Dox did not improve survival in comparison with free doxorubicin. While CP-Dox treatment resulted in elevated intratumoral doxorubicin concentrations in MPNSTs, this increase was absent in UPS tumors. In addition, elevation of CD8+ T cells was observed exclusively in CP-Dox-treated MPNSTs, although these cells were not required for full efficacy of the CP nanoparticle-based chemotherapy. CONCLUSIONS: These results have important implications for treating sarcomas with nanoparticle-encapsulated chemotherapy by highlighting the tumor subtype-dependent nature of therapeutic response.

RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome.

The development of multiple organ dysfunction syndrome (MODS) following infection or tissue injury is associated with increased patient morbidity and mortality. Extensive cellular injury results in the release of nuclear proteins, of which histones are the most abundant, into the circulation. Circulating histones are implicated as essential mediators of MODS. Available anti-histone therapies have failed in clinical trials due to off-target effects such as bleeding and toxicity. Here, we describe a therapeutic strategy for MODS based on the neutralization of histones by chemically stabilized nucleic acid bio-drugs (aptamers). Systematic evolution of ligands by exponential enrichment technology identified aptamers that selectively bind those histones responsible for MODS and do not bind to serum proteins. We demonstrate the efficacy of histone-specific aptamers in human cells and in a murine model of MODS. These aptamers could have a significant therapeutic benefit in the treatment of multiple diverse clinical conditions associated with MODS.

Cancer-Associated Fibroblasts Drive Glycolysis in a Targetable Signaling Loop Implicated in Head and Neck Squamous Cell Carcinoma Progression.

Despite aggressive therapies, head and neck squamous cell carcinoma (HNSCC) is associated with a less than 50% 5-year survival rate. Late-stage HNSCC frequently consists of up to 80% cancer-associated fibroblasts (CAF). We previously reported that CAF-secreted HGF facilitates HNSCC progression; however, very little is known about the role of CAFs in HNSCC metabolism. Here, we demonstrate that CAF-secreted HGF increases extracellular lactate levels in HNSCC via upregulation of glycolysis. CAF-secreted HGF induced basic FGF (bFGF) secretion from HNSCC. CAFs were more efficient than HNSCC in using lactate as a carbon source. HNSCC-secreted bFGF increased mitochondrial oxidative phosphorylation and HGF secretion from CAFs. Combined inhibition of c-Met and FGFR significantly inhibited CAF-induced HNSCC growth in vitro and in vivo (P < 0.001). Our cumulative findings underscore reciprocal signaling between CAF and HNSCC involving bFGF and HGF. This contributes to metabolic symbiosis and a targetable therapeutic axis involving c-Met and FGFR.Significance: HNSCC cancer cells and CAFs have a metabolic relationship where CAFs secrete HGF to induce a glycolytic switch in HNSCC cells and HNSCC cells secrete bFGF to promote lactate consumption by CAFs. Cancer Res; 78(14); 3769-82. ©2018 AACR.

Rapid and Sensitive Detection of Breast Cancer Cells in Patient Blood with Nuclease-Activated Probe Technology.

A challenge for circulating tumor cell (CTC)-based diagnostics is the development of simple and inexpensive methods that reliably detect the diverse cells that make up CTCs. CTC-derived nucleases are one category of proteins that could be exploited to meet this challenge. Advantages of nucleases as CTC biomarkers include: (1) their elevated expression in many cancer cells, including cells implicated in metastasis that have undergone epithelial-to-mesenchymal transition; and (2) their enzymatic activity, which can be exploited for signal amplification in detection methods. Here, we describe a diagnostic assay based on quenched fluorescent nucleic acid probes that detect breast cancer CTCs via their nuclease activity. This assay exhibited robust performance in distinguishing breast cancer patients from healthy controls, and it is rapid, inexpensive, and easy to implement in most clinical labs. Given its broad applicability, this technology has the potential to have a substantive impact on the diagnosis and treatment of many cancers.