A pilot study of multi-modal pain management for same-day discharge after minimally invasive repair of pectus excavatum (Nuss procedure) in children.

BACKGROUND: Despite advancements in minimally invasive repair of pectus excavatum (MIRPE), Nuss procedure, postoperative pain control remains challenging. This report covers a multimodal regimen using bilateral single-shot paravertebral block (PVB) and bilateral thoracoscopic intercostal nerve (T3-T7) cryoablation, leading to significant reduction in length of stay (LOS) and high rate of same-day discharge. METHODS: This is a comparative study of pain management protocols for patients undergoing the Nuss procedure at a single center from 2016 through 2020. All patients underwent the the same surgical technique for the treatment of pectus excavatum at a single center. Patients received bilateral PVB with continuous infusion (Group 1, n = 12), bilateral PVB with infusion and right-side cryoablation (Group 2, n = 9), or bilateral single-shot PVB and bilateral cryoablation (Group 3, n = 17). The primary outcome was LOS with focus on same-day discharge, and the secondary outcome was decreased opioid usage. RESULTS: Eleven of 17 patients in Group 3 (65%) (bilateral single-shot PVB and bilateral cryoablation) were discharged the same day as surgery. The remaining Group 3 patients were discharged the following day with no complications or interventions. Compared to Group 1 (no cryoablation), Group 3 had shorter LOS (median 4.4 days vs. 0.7 days, respectively, p < 0.001) and significantly decreased median opioid use on the day of surgery (0.92 mg/kg vs. 0.47 mg/kg, p = 0.006). CONCLUSION: Findings demonstrate the feasibility of multimodal pain management for same-day discharge after the Nuss procedure. Future multisite studies are needed to investigate the superiority of this approach to established methods. LEVEL OF EVIDENCE: III.

Multicenter Assessment of Cryoanalgesia Use in Minimally Invasive Repair of Pectus Excavatum: A 20-center Retrospective Cohort Study.

OBJECTIVE: To assess the clinical implications of cryoanalgesia for pain management in children undergoing minimally invasive repair of pectus excavatum (MIRPE). BACKGROUND: MIRPE entails significant pain management challenges, often requiring high postoperative opioid use. Cryoanalgesia, which blocks pain signals by temporarily ablating intercostal nerves, has been recently utilized as an analgesic adjunct. We hypothesized that the use of cryoanalgesia during MIRPE would decrease postoperative opioid use and length of stay (LOS). MATERIALS AND METHODS: A multicenter retrospective cohort study of 20 US children's hospitals was conducted of children (age below 18 years) undergoing MIRPE from January 1, 2014, to August 1, 2019. Differences in total postoperative, inpatient, oral morphine equivalents per kilogram, and 30-day LOS between patients who received cryoanalgesia versus those who did not were assessed using bivariate and multivariable analysis. P value <0.05 is considered significant. RESULTS: Of 898 patients, 136 (15%) received cryoanalgesia. Groups were similar by age, sex, body mass index, comorbidities, and Haller index. Receipt of cryoanalgesia was associated with lower oral morphine equivalents per kilogram (risk ratio=0.43, 95% confidence interval: 0.33-0.57) and a shorter LOS (risk ratio=0.66, 95% confidence interval: 0.50-0.87). Complications were similar between groups (29.8% vs 22.1, P =0.07), including a similar rate of emergency department visit, readmission, and/or reoperation. CONCLUSIONS: Use of cryoanalgesia during MIRPE appears to be effective in lowering postoperative opioid requirements and LOS without increasing complication rates. With the exception of preoperative gabapentin, other adjuncts appear to increase and/or be ineffective at reducing opioid utilization. Cryoanalgesia should be considered for patients undergoing this surgery.

Caveolae-Mediated Extracellular Vesicle (CMEV) Signaling of Polyvalent Polysaccharide Vaccination: A Host-Pathogen Interface Hypothesis.

We published a study showing that improvement in response to splenectomy associated defective, in regards to the antibody response to Pneumovax® 23 (23-valent polysaccharides, PPSV23), can be achieved by splenocyte reinfusion. This study triggered a debate on whether and how primary and secondary immune responses occur based on humoral antibody responses to the initial vaccination and revaccination. The anti-SARS-CoV-2 vaccine sheds new light on the interpretation of our previous data. Here, we offer an opinion on the administration of the polyvalent polysaccharide vaccine (PPSV23), which appears to be highly relevant to the primary vaccine against SARS-CoV-2 and its booster dose. Thus, we do not insist this is a secondary immune response but an antibody response, nonetheless, as measured through IgG titers after revaccination. However, we contend that we are not sure if these lower but present IgG levels against pneumococcal antigens are clinically protective or are equally common in all groups because of the phenomenon of "hyporesponsiveness" seen after repeated polysaccharide vaccine challenge. We review the literature and propose a new mechanism-caveolae memory extracellular vesicles (CMEVs)-by which polysaccharides mediate prolonged and sustained immune response post-vaccination. We further delineate and explain the data sets to suggest that the dual targets on both Cav-1 and SARS-CoV-2 spike proteins may block the viral entrance and neutralize viral load, which minimizes the immune reaction against viral attacks and inflammatory responses. Thus, while presenting our immunological opinion, we answer queries and responses made by readers to our original statements published in our previous work and propose a hypothesis for all vaccination strategies, i.e., caveolae-mediated extracellular vesicle-mediated vaccine memory.

Molecular Characterization of Differentiated-Resistance MSC Subclones by Single-Cell Transcriptomes.

Background: The mechanism of tumorigenicity potentially evolved in mesenchymal stem cells (MSCs) remains elusive, resulting in inconsistent clinical application efficacy. We hypothesized that subclones in MSCs contribute to their tumorgenicity, and we approached MSC-subclones at the single-cell level. Methods: MSCs were cultured in an osteogenic differentiation medium and harvested on days 12, 19, and 25 for cell differentiation analysis using Alizarin Red and followed with the single-cell transcriptome. Results: Single-cell RNA-seq analysis reveals a discrete cluster of MSCs during osteogenesis, including differentiation-resistant MSCs (DR-MSCs), differentiated osteoblasts (DO), and precursor osteoblasts (PO). The DR-MSCs population resembled cancer initiation cells and were subjected to further analysis of the yes associated protein 1 (YAP1) network. Verteporfin was also used for YAP1 inhibition in cancer cell lines to confirm the role of YAP1 in MSC--involved tumorigenicity. Clinical data from various cancer types were analyzed to reveal relationships among YAP1, OCT4, and CDH6 in MSC--involved tumorigenicity. The expression of cadherin 6 (CDH6), octamer-binding transcription factor 4 (OCT4), and YAP1 expression was significantly upregulated in DR-MSCs compared to PO and DO. YAP1 inhibition by Verteporfin accelerated the differentiation of MSCs and suppressed the expression of YAP1, CDH6, and OCT4. A survey of 56 clinical cohorts revealed a high degree of co-expression among CDH6, YAP1, and OCT4 in various solid tumors. YAP1 inhibition also down-regulated HeLa cell viability and gradually inhibited YAP1 nuclear localization while reducing the transcription of CDH6 and OCT4. Conclusions: We used single-cell sequencing to analyze undifferentiated MSCs and to discover a carcinogenic pathway in single-cell MSCs of differentiated resistance subclones.

Remote ischemic conditioning in necrotizing enterocolitis: study protocol of a multi-center phase II feasibility randomized controlled trial.

PURPOSE: Remote ischemic conditioning (RIC) is a maneuver involving brief cycles of ischemia reperfusion in an individual's limb. In the early stage of experimental NEC, RIC decreased intestinal injury and prolonged survival by counteracting the derangements in intestinal microcirculation. A single-center phase I study demonstrated that the performance of RIC was safe in neonates with NEC. The aim of this phase II RCT was to evaluate the safety and feasibility of RIC, to identify challenges in recruitment, retainment, and to inform a phase III RCT to evaluate efficacy. METHODS: RIC will be performed by trained research personnel and will consist of four cycles of limb ischemia (4-min via cuff inflation) followed by reperfusion (4-min via cuff deflation), repeated on two consecutive days post randomization. The primary endpoint of this RCT is feasibility and acceptability of recruiting and randomizing neonates within 24 h from NEC diagnosis as well as masking and completing the RIC intervention. RESULTS: We created a novel international consortium for this trial and created a consensus on the diagnostic criteria for NEC and protocol for the trial. The phase II multicenter-masked feasibility RCT will be conducted at 12 centers in Canada, USA, Sweden, The Netherlands, UK, and Spain. The inclusion criteria are: gestational age < 33 weeks, weight ≥ 750 g, NEC receiving medical treatment, and diagnosis established within previous 24 h. Neonates will be randomized to RIC (intervention) or no-RIC (control) and will continue to receive standard management of NEC. We expect to recruit and randomize 40% of eligible patients in the collaborating centers (78 patients; 39/arm) in 30 months. Bayesian methods will be used to combine uninformative prior distributions with the corresponding observed proportions from this trial to determine posterior distributions for parameters of feasibility. CONCLUSIONS: The newly established NEC consortium has generated novel data on NEC diagnosis and defined the feasibility parameters for the introduction of a novel treatment in NEC. This phase II RCT will inform a future phase III RCT to evaluate the efficacy and safety of RIC in early-stage NEC.

Not All that Strictures Is IBD: Gastric Heterotopia Leading to Perforation and Small Bowel Obstruction Mimicking IBD.

A 14-year-old male presented with worsening chronic intermittent abdominal pain, mild anemia, positive fecal occult blood test, and elevated calprotectin. Computerized tomography and magnetic resonance imaging showed ileal dilation with mucosal enhancement and inflammatory changes suspicious for inflammatory bowel disease (IBD). Prominent mucosal folds were suggestive of gastric heterotopia, but Meckel's scan was negative. Upper endoscopy, colonoscopy, and double balloon enteroscopy were grossly and microscopically normal. Laparotomy revealed 17 cm of a dense, inflamed, stenotic segment of ileum. The strictured ileum had perforated and had been concealed by an adjacent loop of small bowel, ultimately producing an obstructive IBD-like picture, but was found to be histologically consistent with gastric oxyntic mucosa. This case illustrates the challenges of diagnosing and treating heterotopic gastric mucosa, and the importance of considering diagnoses other than IBD when evaluating stricturing disease of the small bowel.

Evidence for AJUBA-catenin-CDH4-linked differentiation resistance of mesenchymal stem cells implies tumorigenesis and progression of head and neck squamous cell carcinoma: a single-cell transcriptome approach.

An increasing number of reports indicate that mesenchymal stem cells (MSCs) play an essential role in promoting tumorigenesis and progression of head and neck squamous cell carcinoma (HNSCC). However, the molecular mechanisms underlying this process remain unclear. Using the MSC model system, this study analyzes the molecular pathway by which differentiation resistant MSCs promote HNSCC. MSCs were cultured in osteogenic differentiation media and harvested on days 12 and 19. Cells were stained for cell differentiation analysis using Alizarin Red. The osteogenesis-resistant MSCs (OR-MSCs) and MSC-differentiation-derived osteoblasts (D-OSTBs) were identified and subjected to the single-cell transcriptome analysis. Gene-specific analyses of these two sub-populations were performed for the patterns of differential expression. A total of 1 780 differentially expressed genes were determined to distinguish OR-MSCs significantly from D-OSTB. Notably, AJUBA, ß-catenin, and CDH4 expression levels were upregulated considerably within the OR-MSCs compared to D-OSTBs. To confirm their clinical relevance, a survey of a clinical cohort revealed a high correlation among the expression levels of AJUBA, ß-catenin and CDH4. The results shed new light that OR-MSCs participate in the development of HNSCC via a pathway mediated by AJUBA, ß-catenin, CDH4, and CTNNB1, thereby implying that MSC-based therapy is a promising therapeutic approach in the management of HNSCC.

Opportunities for Regulatory Changes to Promote Pediatric Device Innovation in the United States: Joint Recommendations From Pediatric Innovator Roundtables.

OBJECTIVE: The purpose of this report is to provide insight from pediatric stakeholders with a shared desire to facilitate a revision of the current United States regulatory pathways for the development of pediatric healthcare devices. METHODS: On August 5, 2020, a group of innovators, engineers, professors and clinicians met to discuss challenges and opportunities for the development of new medical devices for pediatric health and the importance of creating a regulatory environment that encourages and accelerates the research and development of such devices. On January 6, 2021, this group joined regulatory experts at a follow-up meeting. RESULTS: One of the primary issues identified was the need to present decision-makers with opportunities that change the return-on-investment balance between adult and pediatric devices to promote investment in pediatric devices. DISCUSSION/CONCLUSION: Several proposed strategies were discussed, and these strategies can be divided into two broad categories: 1. Removal of real and perceived barriers to pediatric device innovation; 2. Increasing incentives for pediatric device innovation.

Autologous Splenocyte Reinfusion Improves Antibody-Mediated Immune Response to the 23-Valent Pneumococcal Polysaccharide-Based Vaccine in Splenectomized Mice.

Common clinical options, currently, for necessary splenectomy are vaccinations and antibiotic prophylaxis. However, despite these two adjuncts, there still occur numerous cases of overwhelming post-splenectomy infection. To examine whether reperfusion of critical splenic lymphocytes could boost immune response, we harvested splenic lymphocytes, reperfused the autologous lymphocytes, and then administered a pneumococcal vaccine (PNEUMOVAX®23, i.e., PPSV23) in splenectomized mice. We found that splenectomy impaired the immune response in the splenectomized group compared to the non-splenectomized group; the splenectomized group with lymphocyte reinfusion had a higher response to polysaccharide vaccination based on antibody titer than the splenectomized group without lymphocyte reinfusion. The sham group with the native spleen had the most elevated antibody titer against the PPSV23 polysaccharide antigen. This may be additive, resulting from contributions of the splenic structure, along with the phagocytic function of the spleen and its constituent cells affecting the antibody response. Reinfusion of splenic lymphocytes may enhance immunity without the complications associated with splenic fragment autotransplantation, which never gained acceptance. This technique is safe and simple since the splenic lymphocytes are autologous and, therefore, not self-reactive, and very similar to autologous blood transfusion. This concept may be beneficial in cases of unavoidable splenectomy, especially in pediatric cases.

Novel Human NKCC1 Mutations Cause Defects in Goblet Cell Mucus Secretion and Chronic Inflammation.

BACKGROUND & AIMS: Infections resulting from intestinal yeast and bacteria affect a large number of patients with deficits in absorptive or secretory epithelial transport mechanisms. The basolateral Na+-K+-2Cl- cotransporter (NKCC1) has been implicated in intestinal epithelial fluid secretion. Two patients with deleterious heterozygous (NKCC1-DFX, DFX for Asp-Phe-stop codon) or homozygous (Kilquist) mutations in SLC12A2 (NKCC1) suffered from gastrointestinal deficits. Because of chronic infections, the colon and the small intestine of the NKCC1-DFX patient were resected surgically. METHODS: To investigate how NKCC1 affects the integrity and function of the gut epithelia, we used a mouse model recapitulating the NKCC1-DFX patient mutation. Electron microscopy and immunostaining were used to analyze the integrity of the colonic mucus layers and immune cell infiltration. Fluorescence in situ hybridization was performed on the distal colon sections to measure bacteria translocation to the mucosa and submucosa. Citrobacter rodentium was used to measure mouse ability to clear enteric infection. A multiplex cytokine assay was used to analyze mouse inflammatory response to infection. RESULTS: We show that NKCC1-DFX expression causes defective goblet cell mucus granule exocytosis, leading to secretion of intact granules into the lumen of the large intestine. In addition, NKCC1-DFX colon submucosal glands secrete mucus that remained attached to the epithelium. Importantly, expression of the mutant NKCC1 or complete loss of NKCC1 function leads to aggravated inflammatory response to C rodentium infection. Compared with wild-type, NKCC1-DFX mice showed decreased expression of claudin-2, a tight junction protein involved in paracellular Na+ and water transport and enteric infection clearance. CONCLUSIONS: Our data indicate that NKCC1-DFX impairs gut barrier function by affecting mucus secretion and immune properties.

Precision Technique for Splenectomy Limits Mouse Stress Responses for Accurate and Realistic Measurements for Investigating Inflammation and Immunity.

Splenectomy in an animal model requires a standardized technique utilizing best practice to avoid variability which can result in adverse impact to the animal resulting in flawed physiologic responses simply due to technique rather than to the studied variables. In the case of the spleen, often investigators are analyzing the animal immune or inflammatory responses. Surgical splenectomy involves many variables from the training and expertise of the surgeon, which directly correlates to surgical technique to the length of operation and ease of the procedure. This operation, in turn, impacts blood loss and insensible fluid losses, sterile technique, unintended trauma to the spleen and surrounding organs, the length of the incision and the duration of the operation with more prolonged exposure to anesthetic agents. All these variables ultimately play a significant role in the experiment since they directly affect the response of the model in terms of inflammation, immune activation, or even suppression. Undesired variables such as these go unnoticed and lead to inaccurate and misleading data.

Single-cell transcriptomes reveal the mechanism for a breast cancer prognostic gene panel.

The clinical benefits of the MammaPrint® signature for breast cancer is well documented; however, how these genes are related to cell cycle perturbation have not been well determined. Our single-cell transcriptome mapping (algorithm) provides details into the fine perturbation of all individual genes during a cell cycle, providing a view of the cell-cycle-phase specific landscape of any given human genes. Specifically, we identified that 38 out of the 70 (54%) MammaPrint® signature genes are perturbated to a specific phase of the cell cycle. The MammaPrint® signature panel derived its clinical prognosis power from measuring the cell cycle activity of specific breast cancer samples. Such cell cycle phase index of the MammaPrint® signature suggested that measurement of the cell cycle index from tumors could be developed into a prognosis tool for various types of cancer beyond breast cancer, potentially improving therapy through targeting a specific phase of the cell cycle of cancer cells.

Outcomes of infants with congenital diaphragmatic hernia treated with venovenous versus venoarterial extracorporeal membrane oxygenation: A propensity score approach.

PURPOSE: Previous studies comparing extracorporeal membrane oxygenation (ECMO) modality for congenital diaphragmatic hernia (CDH) have not accounted for confounding by indication. We therefore hypothesized that using a propensity score (PS) approach to account for selection bias may identify outcome differences based on ECMO modality for infants with CDH. METHODS: We utilized ELSO Registry data (2000-2016). Patients with CDH were divided to either venoarterial (VA) or venovenous (VV) ECMO. Patients were matched by PS to control for nonrandom treatment assignment. Subgroup analyses were conducted based on timing of CDH repair relative to ECMO. Primary analysis was the "intent-to-treat" cohort based on the initial ECMO mode. Mortality was the primary outcome, and severe neurologic injury (SNI) was a secondary outcome. RESULTS: PS matching (3:1) identified 3304 infants (VA = 2470, VV = 834). In the main group, mortality was not different between VA and VV ECMO (OR = 1.01, 95% CI: 0.86-1.18) and there was no difference in SNI between VA and VV (OR = 0.80; 95% CI: 0.63-1.01). For the pre-ECMO CDH repair subgroup, 175 VA cases were matched to 70 VV. In these neonates, mortality was higher for VV compared to VA (OR = 2.10, 95% CI: 1.19-3.69), without any difference in SNI (OR = 1.48; 95% CI: 0.59-3.71). For the subgroup that did not have pre-ECMO CDH repair, 2030 VA cases were matched to 683 VV cases. In this subgroup, VV was associated with 27% lower risk of SNI relative to VA (OR = 0.73, 95% CI: 0.56-0.95) without any difference in mortality (OR = 0.94, 95% CI: 0.79-1.11). CONCLUSION: This study revalidates that ECMO mode does not significantly affect mortality or SNI in infants with CDH. In the subset of infants who require pre-ECMO CDH repair, VA favors survival, whereas, in the subgroup of infants that did not have pre-ECMO CDH repair, VV favors lower rates of SNI. We conclude that neither mode appears consistently superior across all situations, and clinical judgment should remain a multifactorial decision. LEVEL OF EVIDENCE: Level III.

Relapse pathway of glioblastoma revealed by single-cell molecular analysis.

Glioblastoma multiforme (GBM) remains an incurable brain tumor. The highly malignant behavior of GBM may, in part, be attributed to its intraclonal genetic and phenotypic diversity (subclonal evolution). Identifying the molecular pathways driving GBM relapse may provide novel, actionable targets for personalized diagnosis, characterization of prognosis and improvement of precision therapy. We screened single-cell transcriptomes, namely RNA-seq data of primary and relapsed GBM tumors from a patient, to define the molecular profile of relapse. Characterization of hundreds of individual tumor cells identified three mutated genes within single cells, involved in the RAS/GEF GTP-dependent signaling pathway. The identified molecular pathway was further verified by meta-analysis of RNA-seq data from more than 3000 patients. This study showed that single-cell molecular analysis overcomes the inherent heterogeneity of bulk tumors with respect to defining tumor subclonal evolution relevant to GBM relapse.

Microfluidic enrichment of plasma cells improves treatment of multiple myeloma.

Cytogenetic alterations form the basis for risk stratification for multiple myeloma (MM) and guide the selection of therapy; however, current pathology assays performed on bone marrow samples can produce false-negatives due to the unpredictable distribution and rarity of MM cells. Here, we report on a microfluidic device used to facilitate CD45 depletion to enhance the detection of cytogenetic alterations in plasma cells (PCs). Bone marrow samples from 48 patients with MM were each divided into two aliquots. One aliquot was subjected to classic flow cytometry and fluorescent in situ hybridization (FISH). The other first went through CD45+ cell depletion, further enriched by microfluidic size selection. The enriched samples were then analyzed using flow cytometry and FISH and compared to those analyzed using the classic method only. Unlike the traditional method, the microfluidic device removed the CD45+ leukocytes and specifically selected PCs from the remaining white blood cells. Therefore, the microfluidic method (MF-CD45-TACs) significantly increased the percentage of CD38+ /CD138+ cells to 37.7 ± 20.4% (P < 0.001) from 10.3 ± 8.5% in bone marrow. After the MF-CD45-TAC enrichment, the detection rate of IgH rearrangement, del(13q14), del(17p), and 1q21 gains, rose to 56.3% (P < 0.001), 37.5% (P < 0.001), 22.9% (P < 0.001), and 41.7% (P = 0.001), respectively; all rates of detection were significantly increased compared to the classically analyzed samples. In this clinical trial, this microfluidic-assisted assay provided a precise detection of cytogenetic alterations in PCs and improved clinical outcomes.

Spatiotemporal switching signals for cancer stem cell activation in pediatric origins of adulthood cancer: Towards a watch-and-wait lifetime strategy for cancer treatment.

Pediatric origin of cancer stem cell hypothesis holds great promise and potential in adult cancer treatment, however; the road to innovation is full of obstacles as there are plenty of questions left unanswered. First, the key question is to characterize the nature of such stem cells (concept). Second, the quantitative imaging of pediatric stem cells should be implemented (technology). Conceptually, pediatric stem cell origins of adult cancer are based on the notion that plasticity in early life developmental programming evolves local environments to cancer. Technologically, such imaging in children is lacking as all imaging is designed for adult patients. We postulate that the need for quantitative imaging to measure space-time changes of plasticity in early life developmental programming in children may trigger research and development of the imaging technology. Such quantitative imaging of pediatric origin of adulthood cancer will help develop a spatiotemporal monitoring system to determine cancer initiation and progression. Clinical validation of such speculative hypothesis-that cancer originates in a pediatric environment-will help implement a wait-and-watch strategy for cancer treatment.

Tissue Elasticity Bridges Cancer Stem Cells to the Tumor Microenvironment Through microRNAs: Implications for a "Watch-and-Wait" Approach to Cancer.

BACKGROUND: Targeting the tumor microenvironment (TME) through which cancer stem cells (CSCs) crosstalk for cancer initiation and progression, may open new treatments different from those centered on the original hallmarks of cancer genetics thereby implying a new approach for suppression of TME driven activation of CSCs. Cancer is dynamic, heterogeneous, evolving with the TME and can be influenced by tissue-specific elasticity. One of the mediators and modulators of the crosstalk between CSCs and mechanical forces is miRNA, which can be developmentally regulated, in a tissue- and cellspecific manner. OBJECTIVE: Here, based on our previous data, we provide a framework through which such gene expression changes in response to external mechanical forces can be understood during cancer progression. Recognizing the ways mechanical forces regulate and affect intracellular signals with applications in cancer stem cell biology. Such TME-targeted pathways shed new light on strategies for attacking cancer stem cells with fewer side effects than traditional gene-based treatments for cancer, requiring a "watchand- wait" approach. We attempt to address both normal brain microenvironment and tumor microenvironment as both works together, intertwining in pathology and physiology - a balance that needs to be maintained for the "watch-and-wait" approach to cancer. CONCLUSION: This review connected the subjects of tissue elasticity, tumor microenvironment, epigenetic of miRNAs, and stem-cell biology that are very relevant in cancer research and therapy. It attempts to unify apparently separate entities in a complex biological web, network, and system in a realistic and practical manner, i.e., to bridge basic research with clinical application.

Tissue elasticity regulated tumor gene expression: implication for diagnostic biomarkers of primitive neuroectodermal tumor.

BACKGROUND: The tumor microenvironment consists of both physical and chemical factors. Tissue elasticity is one physical factor contributing to the microenvironment of tumor cells. To test the importance of tissue elasticity in cell culture, primitive neuroectodermal tumor (PNET) stem cells were cultured on soft polyacrylamide (PAA) hydrogel plates that mimics the elasticity of brain tissue compared with PNET on standard polystyrene (PS) plates. We report the molecular profiles of PNET grown on either PAA or PS. METHODOLOGY/PRINCIPAL FINDINGS: A whole-genome microarray profile of transcriptional expression between the two culture conditions was performed as a way to probe effects of substrate on cell behavior in culture. The results showed more genes downregulated on PAA compared to PS. This led us to propose microRNA (miRNA) silencing as a potential mechanism for downregulation. Bioinformatic analysis predicted a greater number of miRNA binding sites from the 3' UTR of downregulated genes and identified as specific miRNA binding sites that were enriched when cells were grown on PAA-this supports the hypothesis that tissue elasticity plays a role in influencing miRNA expression. Thus, Dicer was examined to determine if miRNA processing was affected by tissue elasticity. Dicer genes were downregulated on PAA and had multiple predicted miRNA binding sites in its 3' UTR that matched the miRNA binding sites found enriched on PAA. Many differentially regulated genes were found to be present on PS but downregulated on PAA were mapped onto intron sequences. This suggests expression of alternative polyadenylation sites within intron regions that provide alternative 3' UTRs and alternative miRNA binding sites. This results in tissue specific transcriptional downregulation of mRNA in humans by miRNA. We propose a mechanism, driven by the physical characteristics of the microenvironment by which downregulation of genes occur. We found that tissue elasticity-mediated cytokines (TGFß2 and TNFα) signaling affect expression of ECM proteins. CONCLUSIONS: Our results suggest that tissue elasticity plays important roles in miRNA expression, which, in turn, regulate tumor growth or tumorigenicity.