46.15 Tension Influences Small ncRNA Regulatory Landscape of MSC-derived Exosomes During Wound Healing

Posted on January 31, 2019

D. Colchado1, H. V. Vangapandu1, N. Templeman1, H. Li1, Y. Ning1, A. Blum1, P. Bollyky2, S. Keswani1, M. Robertson1, C. Coarfa1, S. Balaji1  1Baylor College Of Medicine,Houston, TX, USA 2Stanford University,Palo Alto, CA, USA

Introduction: Mesenchymal stem cells (MSCs) have a huge therapeutic potential in wound healing. While it is known that the extracellular environment affects the MSC secretome, the role of mechanical tension on the bioactive extracellular vesicles, namely exosomes, released by MSCs, is not known. We hypothesized that mechanical tension regulates MSC exosome production and influences wound healing via paracrine effects on dermal fibroblasts.

Methods: Human MSCs were cultured on silicone membranes +/-10% tonic strain for 24h and analyzed for phenotypic changes (morphology, alpha-SMA, and fibrosis PCR-array) and genes important in exosome biogenesis  (RAb27a-b;SMPD3). Exosomes were isolated and analyzed for size and quantity (Zetasizer). The exosome protein level was quantified (BCA Assay) and Westernblotting (CD63,HSP70,CD9) and Next-Gen Sequencing were performed. Exosomes were labeled by Exo-Glow before use in a primary human dermal fibroblast (FB) migration assay. p-values by ANOVA; (n=3/group).

Results:Tension resulted in the loss of the characteristic morphology of the MSC spindle shape and increased alpha-SMA staining in MSCs. There was a significant change (>2-fold) in ~ 30/77 fibrotic genes with tension. Tension upregulated the expression of IL-10 and IL13RA2, which are also involved in anti-inflammatory cytokine processes. In contrast, pro-fibrotic and pro-inflammatory genes such as Acta-2 and Ccl-2 were downregulated by tension. Additionally, genes encoding fibrinolytic enzymes such as PLAT and growth factors (EGF, VEGFA, and CTGF) were downregulated under tension. Tension downregulated the expression of both RAb27a-b (p<0.01) in MSCs, and there was a corresponding phenotype of perinuclear reorganization of exosomes with an increase in size distribution and protein levels under tension (p<0.05). The three exosome surface markers were verified by Western blotting. Exosomes were enriched for small RNAs as expected. The abundance of tRNA was increased, whereas the miRNAs and lincRNAs in the MSC-derived exosomes were reduced under tension. KEGG analysis of the gene targets and pathways of down regulated miRNAs showed enrichment of intracellular and extracellular wound healing processes. Interestingly, MSC-derived exosomes under static conditions slowed the migration of FB in a scratch wound assay, whereas those derived under tension increased FB migration (p<0.05), but there was no effect of the complete MSC-conditioned media from either static or tension conditions on FB migration.

Conclusion:Mechanical tension induces a fibrogenic and inflammatory phenotype in MSCs. Given that the production and composition of bioactive cargo in MSC exosomes is regulated by tension and can influence FB behavior, we propose that MSC-derived exosomes are a likely target for extracellular communication during wound healing. These insights provide a key role in the development of exosome-based clinical therapies in the context of wound healing and fibrosis.

46.14 A Scar Scoring Method to Predict Recurrence of Discrete Subaortic Stenosis After Surgical Resection

Posted on January 31, 2019

Y. NING1, M. Fahrenholtz1, K. Brown2, E. Steen1, M. Kang2, L. Masri1, L. Wadhwa1, S. Balaji1, K. Grande-Allen2, S. Keswani1  1Texas Children’s Hospital,Pediatrics,Houston, TX, USA 2Rice University,Bioengineering,Houston, TX, USA

Introduction:

Discrete subaortic stenosis (DSS) features the formation of an obstructive fibrous tissue lesion in the left ventricular outflow tract, and while the standard of care is surgical resection, lesions recur in 25-30% of all patients, delineating an “aggressive” phenotype. Here, we define unique histologic parameters that discriminate non-aggressive from aggressive DSS lesions and are consistent with preoperative LVOT gradients, which are known prognostic signs of recurrence.

Methods:

Human DSS patient tissues (n=7) were obtained from the TCH Congenital Heart Biorepository in accordance with IRB protocols. We compared tissue samples from 4 non-aggressive and 3 aggressive recurrent lesions from DSS patients by immunohistochemical staining of cardiac fibroblast DDR2. Next, cell density (H&E), mature/immature collagen ratio (Herovici), and percent DDR2+ cells/HPF were quantified. Lastly, each variable tested was normalized to the ranged set and then combined to generate a DSS aggressiveness score (DAS). Preoperative echocardiographic reports were obtained for all DSS patients whose tissues were analyzed and assessed for mean LVOT gradient. Data was reported as mean ± standard deviation. Statistical analysis was performed using Student’s t-test in which p ≤ 0.05 values were considered significant.

Results:

A differential DDR2 staining was observed between non-aggressive tissues (36.3±27.4% positive/field area) and aggressive tissues, which had almost no measurable expression of DDR2 (1.94±1.90%, p < 0.05). DSS tissues stained by H&E showed significant differences in cell density between aggressive (130±28 cells/image field) and non-aggressive lesions (50±15 cells/field; p < 0.01). Although we observed high variability in both groups, aggressive tissues had increased immature to mature collagen ratios in comparison to non-aggressive ones. Qualitatively, aggressive tissues were more likely to have thin, highly disorganized collagen bundles, whereas non-aggressive tissues had more and thicker collagen bundles. Overall, there was a significant DAS difference between aggressive and non-aggressive tissues (1.94 ±0.46 vs 0.59 ±0.48, p < 0.01) in which the score correlated with the recurrence prediction based on LVOT gradient. Our scoring model will be validated by larger patient sample cohorts.

Conclusion:

Our data underscore histologic features that discriminate aggressive from non-aggressive DSS tissues, which can be quantitatively converted to produce score values that correlate with known predictors of recurrence. This score system may provide a useful means to improve monitoring of aggressive DSS patients.

 

 

46.13 Skeletal Muscle Arteriolar Reactivity to Beta Adrenergic Stimuli is Attenuated After Cardiac Surgery

Posted on January 31, 2019

O. Ziegler1, K. Anderson1, Y. Liu1, V. Pavlov1, J. Fingleton1, A. Ehsan1, N. Sodha1, A. Usheva1, J. Feng1, F. Sellke1  1The Warren Alpert Medical School of Brown University,Cardiovascular Research Center,Providence, RI, USA

Introduction: Challenges in blood pressure management during and after cardiac surgery are a significant cause of morbidity and mortality. Previously our lab has found that cardioplegic arrest and cardiopulmonary bypass (CP/CPB) is associated with impaired arteriolar responsiveness to phenylephrine. However, concomitant changes in alpha adrenergic receptor abundance and localization were not observed. Here, we further investigate the role of beta adrenergic mediators in skeletal muscle arterioles pre- and post- CP/CPB as well as beta receptor abundance and localization to arterioles in skeletal muscle.

Methods:  Skeletal muscle arterioles (90-150 µm in diameter) were collected pre- and post- CP/CBP from patients undergoing cardiac surgery (CABG or AVR).  Ex vivo microvascular reactivity was measured in response to the beta adrenergic receptor agonist isoproterenol (concentrations 10-9 to 10-4 M). 

Results: Isoproterenol induced dose-dependent relaxation response in both pre-and post-CP/CPB vessels. Post-CP/CPB vessel relaxation response to isoproterenol was significantly attenuated (p < 0.05 vs pre-CP/CPB). There were no significant changes in localization of beta 1, beta 2, or beta 3 adrenergic receptors to arterioles of skeletal muscle as assayed by immunohistochemistry between these groups. Nor were there significant changes in beta receptor abundance as assayed by western blotting.  

Conclusion: These findings suggest there is diminished peripheral arteriolar responsiveness to beta adrenergic stimuli after CP/CBP, though this is not explained by beta adrenergic receptor abundance or localization. This further suggests that CP/CPB is associated with vasoplegia in the peripherial microvasculature early after cardiac surgery.

 

46.12 Self DNA Drives Inflammasome Activation by STING During Aortic Aneurysm and Dissection Development

Posted on January 31, 2019

Y. Wang1,2, W. Luo1,2, P. Ren1,2, J. Guo1,2, C. Zhang1,2, L. Zhang1,2, J. Coselli1,2,3, Y. Shen1,2,3, S. LeMaire1,2,3  1Baylor College Of Medicine,Division Of Cardiothoracic Surgery,Houston, TX, USA 2Texas Heart Institute,Department Of Cardiovascular Surgery,Houston, TX, USA 3Baylor College Of Medicine,Cardiovascular Research Institute,Houston, TX, USA

Introduction:
During tissue injury, damaged cells activate inflammatory cells, which promote further injury, leading to disease progression. However, the mechanisms underlying inflammation initiation remain to be illustrated. Here, in a setting of aortic aneurysm and dissection (AAD), we show that self DNA from dying smooth muscle cells drive NLRP3 inflammasome activation and inflammatory response by activating pro-inflammatory cytosolic DNA sensor stimulator of interferon genes (STING) and interferon regulatory transcription factor (IRF3) signaling.

Methods:
The activation of NLRP3 inflammasome, STING and IRF3 were examined in macrophages of aortic tissues from patients with sporadic ascending thoracic AAD (ATAAD). The effects of SMC-derived DNA on macrophage activation and the involvement of STING-IRF3 in the NLRP3 inflammasome activation were examined in cultured macrophages. The role of STING in inflammasome activation and aortic inflammation was further examined in Sting-deficient (Sting-/-) mice in a sporadic AAD model induced by a high-fat diet and angiotensin II infusion.

Results:
In macrophages of aortic tissues from patients with ATAAD, we observed significant amount of cytosolic DNA and activation of the STING-IRF3 pathway, which was associated with NLRP3 inflammasome activation. In cultured macrophages, the engulfment of SMC-derived DNA activated STING and IRF3, which in turn induced the activation of NLRP3 inflammasome. In the sporadic AAD mouse model, genetically deleting Sting or pharmacologically inhibiting Sting with inhibitor Amlexanox significantly reduced the challenge-induced NLRP3 inflammasome activation and aortic inflammation.

Conclusion:
DNA from damaged SMCs, by triggering macrophage cytosolic DNA sensor STING-IRF3 pathway, activates NLRP3 inflammasome, leading to aortic inflammation. This STING-IRF3 pathway mediates the cross-talk between vascular damage and the inflammasome activation and inflammation induction.

46.10 A Microfluidic Study of Non-Glycemic Effects of Metformin on Hyperglycemia-stress exposed endothelium

Posted on January 31, 2019

J. V. Martin1, D. M. Liberati1, L. N. Diebel1  1Wayne State University,Surgery/School Of Medicine,Detroit, MI, USA

Introduction: Stress related hyperglycemia is associated with poor outcomes in trauma and critically ill patients. Correction of hyperglycemia may improve clinical outcomes; however tight glycemic control with insulin may lead to hypoglycemic episodes and resultant glucose variability. Metformin has demonstrated efficiency in hyperglycemia treatment in burn and non-diabetic coronary bypass surgery patients. These effects may be partly due to non-glycemic effects including a beneficial effect on endothelial function. We have shown that acute hyperglycemia exacerbates trauma induced endothelial and glycocalyx injury in an in vitro model. We therefore studied the effect of metformin on endothelial injury in a biomimetic model of hemorrhagic shock using a microfluidic device.

Methods: Human umbilical vein endothelial cell (HUVEC) monolayers were established and perfused in a microfluidic device. Perfusion conditions included media alone (control), media + 80 or 200 mg/dl glucose with or without hypoxia-reoxygenation (H/R) + epinephrine (Epi) to mimic the microcirculation following hemorrhagic shock (HS). Metformin (50µM) was added after glucose exposure. Markers of endothelial glycocalyx degradation were syndecan-1 (syn-1) and hyaluronic acid (HLA) shedding. Endothelial cell activation markers included soluble thrombomodulin (sTM), and angiopoietin-1 and 2 concentrations in the perfusate. Reactive oxygen species (ROS) and inducible nitric oxidase synthase (iNOS) generation were measured using fluorescent imaging.

Results: See Table.

Conclusion: Metformin ameliorated stress hyperglycemia effects on glycocalyx and vascular barriers in a biomimetic model of the microcirculation following HS. These effects may be related to decreased ROS and iNOS generation. Microfluidics may be useful to study the endotheliopathy of trauma and HS. 

 

46.08 Role of Plasma Soluble CD154 in Post-Traumatic Systemic Inflammatory Response Syndrome

Posted on January 31, 2019

D. T. Lubkin1,6, M. Song1,6, M. Bishawi1,6, T. V. Brennan2, E. A. Elster3,4,6, A. D. Kirk1,5,6  1Duke University Medical Center,Surgery,Durham, NC, USA 2Cedars-Sinai Medical Center,Surgery,Los Angeles, CA, USA 3Walter Reed Army Medical Center,Washington, DC, USA 4Uniformed Services University Of The Health Sciences,Bethesda, MD, USA 5Duke University Medical Center,Immunology,Durham, NC, USA 6Uniformed Services University Of The Health Sciences,Surgical Critical Care Initiative (SC2i),Bethesda, MD, USA

Introduction:  Systemic inflammatory response syndrome (SIRS) is a major cause of delayed morbidity and mortality in trauma patients and can result in hypoperfusion, organ failure, and death. Soluble CD154 (CD40 Ligand) is released by platelets activated in response to endothelial damage and may contribute to systemic inflammation through stimulation of CD40 on monocytes and endothelial cells, leading to release of interleukin-6 (IL-6), IL-8, and monocyte chemotactic protein 1 (MCP-1). The aim of this study was to investigate the association between plasma levels of soluble CD154 (sCD154) in trauma patients and the development of post-traumatic SIRS.

Methods:  Samples and clinical data were prospectively collected from acutely injured patients as part of the Surgical Critical Care Initiative (SC2i). All samples were collected within 24 hr of injury. Plasma sCD154 levels were measured by enzyme-linked immunosorbent assay (ELISA) and serum IL-6, IL-8, and MCP-1 levels were measured by Luminex® assay.

Results: Of the 29 patients included, 15 (51.7%) developed SIRS and 14 (48.3%) did not. Median plasma sCD154 level was significantly higher in the SIRS group than in the no SIRS group [Median (IQR) 72.19 (63.78-116.4) vs. 55.72 (42.07-77.63) pg/ml, p=0.017]. Median serum IL-6 level was significantly higher in the SIRS group [SIRS: 78.73 (38.2-112.6) vs. no SIRS: 34.09 (17.36-47.41) pg/ml, p=0.018]. Median levels of IL-8 [34.45 (21.27-68.5) vs. 23.77 (8.64-54.89) pg/ml, p=0.16] and MCP-1 [508.1 (417.1-854.3) vs. 415.1 (228-680.7) pg/ml, p=0.38] trended higher in the SIRS group, but these differences did not reach statistical significance.

Conclusion: sCD154 levels are elevated in patients with clinical SIRS and thus may be useful as a biomarker, alone or in combination with other inflammatory cytokine levels. Further work is warranted to assess the role of sCD154 in the evolution of post-traumatic inflammatory responses, and its blockade as a potential therapeutic strategy for the prevention and treatment of SIRS.

 

46.07 Elevated Levels of Lipoprotein (a) Induces an Anticoagulant Effect in Whole Blood

Posted on January 31, 2019

M. G. Bartley1, N. G. Vigneshwar1, J. R. Coleman1, J. M. Samuels1, A. Sauaia1, A. Banerjee1, C. Silliman3,4, E. E. Moore1,2  1University Of Colorado Denver,Surgery,Aurora, CO, USA 2Denver Health Medical Center,Surgery,Aurora, CO, USA 3Bonfils Blood Center,Research Laboratory,Denver, CO, USA 4University Of Colorado Denver,Pediatrics,Aurora, CO, USA

Introduction:  Lipoprotein (a) [Lp(a)] is a composite of low-density lipoprotein (LDL), apolipoprotein B-100 (ApoB) and apolipoprotein A [Apo(a)]. Apo(a) contains a serine protease and multiple kringle domains, specifically kringle IV and V that share homology to the fibrinolytic zymogen plasminogen. Previous studies have shown Lp(a) can bind to endothelial cell surfaces inhibiting pericellular plasminogen activation.  The majority of Lp(a) studies have focused on the association between atherosclerotic risk and elevated plasma levels with limited data on coagulative properties. We hypothesize that elevated levels of Lp(a) will induce fibrinolytic shutdown via inhibition of plasminogen.

Methods:  Exogenous Lp(a) low (20mg/dl) and high dose(60mg/dl) physiologic concentrations, tissue plasminogen activator(tPA) were added to whole blood (WB) samples from healthy volunteers(n=8).  Samples were assessed using citrated native thromboelastography(TEG). Fibrinolysis was initiated by adding tPA to samples containing either whole blood alone or with low dose (20mg/dl) or high dose (60mg/dl) of Lp(a). The following TEG measurements were recorded: clot initiation (R time, fibrin polymerization (angle) and clot strength [maximum amplitude (MA) clot lysis time 30 minutes after reaching MA (LY30)].

Results: LY30 increased significantly with the addition of tPA in whole blood(WB 1.2% vs WB+tPA11.7 p=0.002) but was unaffected by low or high dose Lp(a) with tPA (low dose 11.3% p=0.99 vs high dose 7.8% p=0.45).  Low dose Lp(a) did not modify coagulation however, high dose Lp(a) prolonged clot initiation (R time), reduced clot formation (angle) and strength (MA) (WB 14.2min vs low 21.1 p=0.93; WB 14.2min vs high dose 99.0min p=0.0001) (WB 54mm vs low dose 49mm p=0.92; WB 54mm vs high dose 26.9mm p=0.005) (WB 40.8 deg vs low dose 33.4 deg p=0.68; WB 40.8 deg vs 11.35 deg p=0.0002).

Conclusion: Lp(a) did not modify fibrinolysis, but high doses produced un anticipated hypocoagulability.  The mechanism and clinical implications warrant further investigation.

 

46.06 Whole Blood Thrombin Generation: Distinct from Plasma and Unique in Severely Injured Patients

Posted on January 31, 2019

J. R. Coleman1, S. Butenas2, J. M. Samuels1, M. G. Bartley1, N. G. Vigneshwar1, J. T. Nelson4,5, J. J. Ryon3, A. Olson2, A. Sauaia1, A. Banerjee1, C. C. Silliman5, E. E. Moore2,3  1University of Colorado-Denver,Surgery,Aurora, CO, USA 2University of Vermont,Biochemistry,Burlington, VT, USA 3Denver Health,Surgery,Denver, CO, USA 4Rosalind Franklin University of Medicine and Science,Chicago Medical School,North Chicago, IL, USA 5Children’s Hospital Colorado,Hematology,Aurora, CO, USA

Introduction:
Thrombin is a serine protease responsible for conversion of soluble fibrinogen into insoluble fibrin and is the end-point of the coagulation cascade. As such, measurement of thrombin generation (TG) offers an important description of the coagulation status of a patient. Historically, plasma TG has been used to describe coagulopathy of trauma, but the description of whole blood (WB) TG is lacking in the literature. This study aims to compare plasma and WB TG and then to examine clinical correlations with WB TG in trauma patients. We hypothesize that 1) WB TG does not correlate with plasma TG and 2) severely injured trauma patients demonstrate a unique WB TG profile.

Methods:
To compare plasma and whole blood TG, blood was collected from healthy volunteers. WB TG was performed using a prototype point-of-care WB TG device (Near Patient Testing Thrombin Generation; NPT-TG) and the remaining blood was spun down to perform platelet-poor plasma TG with a BioTek plate reader (CAT analogue). To examine clinical correlatives in trauma patients, initial blood was collected as part of a prospective study of trauma activation patients before arrival or upon presentation to a large, urban, level-1 trauma center. WB TG was performed within one hour of venipuncture. TG yields the following measurements: lag time, peak thrombin, time to peak thrombin, maximum rate (or velocity index) and endogenous thrombin potential (ETP). Correlation between plasma and WB TG were compared using Pearson’s correlation. WB TG measurements were compared between severely injured patients (new injury severity score [NISS] > 15) and non-severely injured patients (NISS ≤ 15) with the Mann-Whitney test.

Results:
Overall, 10 healthy volunteers were included in this study. There was poor correlation between the plasma and WB TG, with plasma reflecting a more prolonged time to peak thrombin, higher rate of TG and higher ETP (R2 values ranged from 0.07 to 0.47, p values 0.17 to 0.83). Additionally, 49 trauma patients were included in this study. The average age was 37.6 years and the majority (76%) were male. 59% presented after blunt trauma and the median NISS was 17. On examining WB TG, severely injury patients (NISS>15) had a prolonged lag time (4.3 min versus 3.0 min, p=0.03), depressed peak thrombin (123.7 nM versus 135.6 nM, p=0.02) and slower rate of thrombin generation (37.2 nM/min versus 54.2 nM/min, p=0.02) than less severely injured patients. They also demonstrated a trend towards prolonged time to peak thrombin (7.8 min versus 5.6 min, p=0.08) and decreased ETP (798.7 versus 831.8 nM/L x min, p=0.27).

Conclusion:
Plasma and WB TG do not correlate, supporting the cellular-based understanding of coagulation. These results challenge the previous assumption that severely injured trauma patients are believed to be initially hypercoagulable, leading to consumptive coagulopathy and hypocoagulopathy. These data instead describe an initial hypocoagulable profile which is pronounced after severe injury and characterized by prolonged time to thrombin formation, decreased maximum thrombin and slow rate of thrombin generation.
 

46.05 Major Thermal Burn Injury Results in Elevated Thrombin Activatable Fibrinolysis Inhibitor Activity

Posted on January 31, 2019

R. L. Ball1,2, M. C. Bravo3, T. Orfeo3, L. T. Moffatt1,4, K. E. Brummel-Ziedins3, J. W. Shupp1,2,4,5  1MedStar Health Research Institute,Firefighters’ Burn And Surgical Research Laboratory,Washington, DC, USA 2MedStar Washington Hospital Center,The Burn Center,Washington, DC, USA 3University Of Vermont College Of Medicine / Fletcher Allen Health Care,Department Of Biochemistry,Burlington, VT, USA 4Georgetown University School of Medicine,Department Of Biochemistry,Washington, DC, USA 5Georgetown University School of Medicine,Department Of Surgery,Washington, DC, USA

Introduction: Activated thrombin activatable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase whose primary physiological substrate is plasmin-modified fibrin. TAFIa removes C-terminal lysine and arginine residues generated by plasmin, thus reducing the rate of plasminogen activation, resulting in transient dampening of fibrinolysis. Expression of TAFIa/ai associated with major thermal burn injury (MThBI) has not been previously reported. For the first time, circulating levels of TAFIa/ai following thermal injury were investigated in order to advance the understanding of burn-induced coagulopathy.

Methods:  In a prospective observational study, admission blood samples were collected within 6 hours of injury from 15 patients with MThBI greater than 20% total body surface area (TBSA) per protocol approved by the Institutional Review Board. Citrate plasma was prepared within 15 minutes of blood collection. TAFIa/ai levels were quantified with ELISA. Hemostatic activation in MThBI patients was established by measuring d-dimer and plasmin-antiplasmin complex (PAP). Chart review was conducted to determine demographics, TBSA, and 30-day mortality. Comparisons were made between patients that died (n = 10) versus survived (n = 5). Statistical significance was determined using Mann-Whitney U tests.

Results: In this study, MThBI patients had significantly higher levels of TAFIa/ai (49.8±25.9 ng/mL, range: 18.9-105.4 ng/mL) compared to the mean of 60 healthy individuals (15.3±6.8 ng/mL; p=<0.001). 80% of these patients showed coordinated increases above the upper limit of the normal range for d-dimer, PAP, and TAFIa/ai. Among the group of patients that survived, mean TAFIa/ai was 45.5±29.2 ng/mL compared to 58.4±17.1 in patients who died (p = 0.21).

Conclusion: On admission, MThBI patients have increased expression of TAFIa/ai. This suggests that this group of patients exhibited inhibition of fibrinolysis early after injury coinciding with evidence of hemostatic activation. Taken together, this potentially represents a mechanism to increased clot stability following MThBI.

 

46.04 Validation of D-dimer to Predict Early Hemorrhagic Expansion Following Traumatic Brain Injury

Posted on January 31, 2019

A. Duong1, X. Zhang1, J. Karri2, B. Cotton1, C. Wade1, J. Cardenas1  1McGovern Medical School,Surgery,Houston, TX, USA 2Baylor College Of Medicine,Department Of Physical Medicine And Rehabilitation,Houston, TX, USA

Introduction:
Progressive hemorrhagic injury (PHI), the early expansion of intracranial hemorrhage (ICH) following a traumatic brain injury (TBI), is associated with a five-fold increase in the risk of neurological decline, complications, and mortality. Our lab, and others, had previously demonstrated fibrinolysis to be an important mechanism underlying PHI and that admission levels of D-dimer, a byproduct of fibrinolysis, may be used to screen for PHI. In this study, we aimed to independently validate the previously proposed D-dimer of 3.6 µg/mL threshold for PHI identification and hypothesized it would accurately differentiate patients with PHI from those with stable hemorrhage (SH).

Methods:
This was a single institution, retrospective analysis of prospectively collected data between September 2013 and March 2018. All highest-level trauma activation patients between ages of 18-55 years, with an initial head CT scan at admission showing severe TBI, defined as presence of ICH, and a repeat scan within 6 hours following, were included. This limit on age was set to minimize inclusion of patients on anticoagulants.  Prisoners, pregnant women, and patients with non-survivable head injury were excluded. Differentiability of D-dimer was evaluated by receiver operating curve analysis. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for the previously identified cut point of 3.6 µg/mL.

Results:
From September 2013 to March 2018, 244 patients between 18-55 years old were admitted to Memorial Hermann Hospital with severe TBI.  An additional 75 patients were excluded due to non-survivable injuries, lack of CT scan, did not consent, or plasma was not obtained. This left 169 patients for analysis. Of those, 54% had SH and 46% had PHI. Patients who developed PHI received more red blood cell transfusions (p =0.02) and had significantly fewer ventilator-, intensive care unit-, and hospital-free days compared to SH patients (all p <0.01). Furthermore, PHI patients had significantly higher D-dimer levels than those with SH (8.1 (4.6, 17.1) μg/mL vs 5.7 (1.8, 11.7) μg/mL, respectively, p <0.01). The D-dimer threshold of 3.6 μg/mL yields a sensitivity of 0.83 (95% CI 0.74-0.91), specificity of 0.40 (95% CI 0.30-0.50), positive predictive value of 0.53 (95%CI 0.44-0.62), and negative predictive value of 0.74 (95% CI 0.61-0.86).

Conclusion:
Clinical use of D-dimer on admission in TBI patients could provide a sensitive screening tool to identify patients at high risk for PHI. Such a tool could be used for early administration of life-saving interventions for high risk patients and also eliminate costly, invasive screening procedures for patients at low risk of PHI.

46.03 Adenosine, Lidocaine, Mg2+ as an Adjunct to Whole Blood Resuscitation does not Improve Coagulopathy

Posted on January 31, 2019

K. Wolf1, L. J. Schaub2, C. G. Morgan2, P. Hemond2, J. J. Glaser2  1Wake Forest University School Of Medicine,Winston-Salem, NC, USA 2Naval Medical Research Unit San Antonio,Fort Sam Houston, TX, USA

Introduction:  Acute traumatic coagulopathy is a major contributor to mortality within the first 24 hours of injury. The use of Hextend® as a pre-hospital fluid per military clinical practice guidelines amplifies this life threatening coagulopathy. ALM is a cardioplegic agent that has been shown to mitigate coagulopathy in animal models of traumatic shock. We hypothesized that ALM, as an adjunct to whole blood resuscitation, would improve coagulopathy as compared to whole blood resuscitation alone in a swine model of shock.

Methods:  Swine (N=18) were randomized to three treatment groups: (1) Whole blood alone, (2) Whole blood + 30 mL of ALM bolus (A:1mg/kg; L:2 mg/kg; M:0.5 mg/kg) infused at 5 mL/min, or (3) Whole + 30 mL of vehicle control (VC) (0.9% NaCl bolus) infused at 5 mL/min.  Animals underwent a pressure controlled hemorrhage to MAP of 30 mmHg. At T= 0, 60 min of simulated prehospital care was initiated per TCCC guidelines (Hextend®), followed by 4 hours of ‘hospital care’ resuscitation with varying adjunct treatments above. Physiologic variables were recorded; arterial blood gas and viscoelastometry analyses were performed at specified time points.

Results: Lactate levels were significantly lower in the WB+ALM group compared to the WB+VC groups during shock and resuscitation (T= 0 through T= 120). WB+VC group base deficit was significantly worsened as compared to WB and WB+ALM when treatment began (T= 60). MAP was equivalent between groups except at T= 65, when WB+ALM MAP dropped, and at T=75/85 when WB+ALM MAP increased. All values were equivalent at T= 300. After treatment, T= 60, all groups showed comparable viscoelastometry parameters with the exception of ExTEM MCF at T=180.

Conclusion: In this model, ALM as an adjunct to WB resuscitation demonstrated no advantage to WB alone. While lactate and BD showed improvement during shock and initial resuscitation with ALM, these values were equivalent between WB and WB+ALM at all other time points after resuscitation. No differences were noted in coagulopathy. Previous reports of correction of coagulopathy, in similar animal models, were not reproduced here. Further investigation into the interactions of ALM and whole blood may be warranted to clarify ALM’s effects on coagulopathy. 

 

46.02 Circulating caspase activity in trauma patients as a biomarkers of injury severity and short term outcomes

Posted on January 31, 2019

M. Bishawi1,2, B. Johnston1,2, C. Almond1,2, L. Stempora1,2, D. Bowles1, A. T. Limkakeng1,2, J. Cheeseman1,2, E. Elster2,3,4, A. Kirk1,2  1Duke University Medical Center,Surgery,Durham, NC, USA 2Uniformed Services University (USU) Surgical Critical Care Initiative (SC2i),Bethesda, MD, USA 3Walter Reed National Military Medical Center,Bethesda, MD, USA 4Uniformed Services University of the Health Sciences,Bethesda, MD, USA

Introduction: The caspase family of cysteine proteases play an essential role in the signaling pathways associated with apoptosis and inflammation.  Circulating caspase activity levels may be an early indicator of overall cell death and inflammatory system activation in injured military personnel. We hypothesized that circulating caspase activity levels may correlate with injury severity in trauma patients, and early changes in these levels may predict short term patient outcomes.     

Methods: This was an exploratory pilot sub study from the SC2i initiative. Longitudinal plasma samples derived from 11 trauma patients were used.  The patients were divided into two groups based on their short term outcomes.  Group A had an overall benign hospital stay, and group B were patients having a prolonged hospital stay with significant post injury morbidity and mortality.  Caspase activities in all available plasma samples were measured using a commercially available bioluminescent assay kit (Promega Corporation Maddison WI).  Caspase activity level data were then merged with and compared to clinical data obtained in a prospectively maintained study database. 

Results:Group A patients (n=5) trended to be younger (36.2±21 vs. 58.7±18, p=0.117), had a trend of a higher presenting GCS score (14.6±0.9 vs. 10.2±5.7, p=0.113), and had a lower expanded ISS score (25.2±11 vs. 43.5±14  p=0.03) as compared to Group B patients (n=6).  Overall hospital length of stay was also shorter for Group A patients (4.5±1.9 vs. 33.7±23.7, p=0.03), as well as ICU length of stay (0.5±1 vs. 24.6±19, p=0.03).  All group A patients were discharged home compared to none of group B patients (p<0.01).  In-hospital mortality was significantly higher for Group B patients (67% vs. 0%, p<0.01). Caspase activity levels were similar between the groups early after injury, but significantly increased in group B patients over time while remining unchanged for group A patients.  (Figure)

Conclusion:In this pilot study, circulating caspase activity levels significantly increased in severely injured patients that experienced substantial midterm morbidity and mortality. A larger study including more patients to further explore the utility of caspase activity measurements in risk stratification for injured personnel is warranted. If confirmed, caspase activity measurements may become an important biomarker that will help in post injury patient care and resource utilization to further improve outcomes.  

 

45.20 Heat Shock Protein 70 modulates Dendritic Cell Function in Tumor Microenvironment

Posted on January 31, 2019

B. Giri1, A. Ferrantella1, P. Sharma1, V. Sethi1, S. Modi1, Z. Malchiodi1, B. Garg1, S. Ramakrishnan1, S. Lavania1, S. Banerjee1, A. Saluja1, V. Dudeja1  1University Of Miami,Surgery,Miami, FL, USA

Introduction: Dendritic cells (DCs) are an up and coming technique for immune cell vaccination in tumor control strategies. We evaluated whether absence of heat shock protein 70 in dendritic cells altered their antitumor properties.

 

Methods: Dendritic cells from tumor bearing mice from either WT or HSP70-/- mice were selected by pan dendritic cell isolation kit. Then they were inoculated in mice with WT or HSP70-/- background and then given a tumor challenge of pancreatic cancer with KPC cells. In another experiment, WT or HSP70-/- dendritic cells were exposed to tumor cell lysates derived from the mouse pancreatic cancer cell line, KPC, in vitro.

 

Results: Tumors that were formed in WT mice receiving HSP70-/- DC were smaller than those receiving WT Dendritic cells. Overall vaccination with DC decreased tumor formation rate in both WT and KO mice but the decrease in tumor size was greater when HSP70-/- DC were implanted. Similarly, HSP70-/- dendritic cells had greater expression of anti-tumorigenic MHCII peptide on their surface when they were exposed to tumor lysates from KPC cells suggesting that HSP70 deficient DCs were more adept at mounting an anti-tumor immune effect. 

 

Conclusion: HSP70 modulates dendritic cell function in the immune response against cancer.

45.19 HUMAN MELANOMA CELL LINE SK-MEL 28 EXPRESSES FUNCTIONAL REC PROTEIN

Posted on January 31, 2019

M. Sharma2, P. Jackson3, J. Huang2, D. Grabski1, D. Rekosh2,4, M. Hammarsjkold2,4, S. K. Rasmussen1  1University of Virginia,Surgery,Charlottesville, VA, USA 2University of Virginia,Microbiology, Immunology, And Cancer Biology,Charlottesville, VA, USA 3University of Virginia,Internal Medicine,Charlottesville, VA, USA 4University of Virginia,Thaler Center For HIV And Retrovirus Research,Charlottesville, VA, USA

Introduction: Melanoma is a skin cancer that is responsible for >9,000 deaths In the U.S. yearly; 5-year survival from advanced disease is <40%. Despite advances in the understanding and treatment of this cancer, there are limitations in treatments, and new targets for therapy are needed. More insight into neoantigen expression in melanoma will be beneficial to bringing new innovations to fruition. 
Expression from the youngest class of human endogenous retroviruses (HERV-K) is known to be upregulated in melanoma. HERVs are derived from ancient retroviral infections and they comprise 8% of the genome. The effect of HERV-K upregulation in melanomas is unknown. However, HERV-Ks encode several different proteins that can be expressed in human cells. One of these proteins is the regulatory protein Rec. We probed the function of this protein in melanoma cell lines using a novel Lentiviral GFP reporter.
HERV-K Rec is a nuclear-export factor that exports mRNA with retained introns. Some studies postulate that Rec protein may have oncogenic properties, which makes it a provocative target for investigation. Many of the rec sequences in our genome have been subject to deletions and other inactivating mutations during genomic evolution; it remains to be shown if any of the Rec proteins expressed in melanoma cells are functional.

Methods: To analyze the expression of Rec in the SK-28 Mel cell line, we isolated, amplified, and cloned rec mRNAs expressed in these cells. Multiple cDNA clones were then sequenced. The most commonly detected variants were cloned and tested for functionality. Next, we utilized a lentiviral vector that had been engineered to express green fluorescent protein (GFP) only in the presence of functional Rec protein, to determine if Rec protein produced by melanoma cell lines was functional. We utilized a protein that inactivates Rec (Td-Rev) to eliminate native cellular Rec activity, in order to confirm that the induced GFP expression from our vector was Rec-dependent.

Results:65% of Rec cDNA clones obtained from the SK-28 cells had an open reading frame identical to a previously identified functional Rec, whereas 35% of the clones contained mutations, implying that multiple rec loci were expressed in this cell line. Three rec mutants were tested in transfection assays. One was shown to have decreased activity, whereas the other two were non-functional. We found that the Lentiviral  vector that expresses Rec-dependent GFP showed significant activity in SK-28 cells, indicating expression of functional Rec protein in these cells. When Td-Rev, a protein that inhibits Rec activity, was co-transfected, GFP expression was reduced to background levels.

Conclusion:SK-Mel28 cells express functional Rec protein as well as non-functional Rec proteins from mutated loci. Furthermore, expression of a Rec dependent GFP reporter can be inhibited by targeting Rec. Further studies on the role of Rec in melanoma are warranted, as Rec may be a viable therapeutic target.

 

45.18 The Dual Role of MicroRNA-34a in Breast Cancer

Posted on January 31, 2019

J. C. Sporn1, E. Katsuta1, L. Yan2, K. Takabe1  1Roswell Park Cancer Institute,Surgical Oncology,Buffalo, NY, USA 2Roswell Park Cancer Institute,Biostatistics & Bioinformatics,Buffalo, NY, USA

Introduction:   MicroRNAs are short RNA molecules of about 18 to 24 nucleotides in length that target certain messenger RNAs (mRNAs) leading to its degradation and thereby preventing them from coding for a specific protein. MicroRNA-34a (miR-34a) was found to be lost or decreased in many different cancers, suggesting a role as a tumor suppressor, and re-expression of miR-34a has been investigated as a potential treatment of cancers in clinical trials. In breast cancer models, miR-34a was shown to be implicated in proliferation and cell differentiation and its expression was inhibited within the breast cancer stem cell pool. We were interested in the role of miR-34a in various breast cancer subtypes to further elucidate the context-specific role of miR-34a in cancer.

Methods:   We utilized The Cancer Genome Atlas (TCGA) containing genetic and molecular data, clinical profiles and survival information for 985 breast cancers. Survival analysis was performed comparing a group with low expression of miR-34a to a group with high expression. Expression of miR-34a was compared based on clinicopathological parameters including ER and HER2 status. Gene Set Enrichment Analysis was performed for the miR-34a high and low groups.

Results:  Patients with tumors with low expression of miR-34a have a better prognosis than patients with tumors with high expression of miR-34a (p=0.003). An identical association was found for the ER positive cohort where patients with tumors with low expression of miR-34a had an improved overall survival compared to patients with tumors with high expression of miR-34a (p<0.001). Interestingly, this effect was not only lost, but reversed in the ER negative cohort where high miR-34a expression was associated with improved disease-free survival compared to low miR-34a expression.

Conclusion:  MiR-34a plays complex and even opposing roles in breast cancer. While we confirmed its classical role as a tumor suppressor in the ER negative cohort, we demonstrated an oncogenic role in ER positive cancers where high expression was associated with a worse prognosis.

45.17 Expression of Long Non-Coding RNA in Colon Adenocarcinoma and Matched Normal Adjacent Epithelium

Posted on January 31, 2019

C. L. Bishop1, S. O’Brien1, L. Dailey1, V. Stephen1, M. Eichenberger1, S. Galandiuk1  1University of Louisville School of Medicine,Price Institute of Surgical Research,Louisville, KY, USA

Introduction: Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths in the U.S. Long non-coding RNAs (lncRNAs) are large (>200 nucleotides) RNA molecules that make up a significant portion of the non-coding transcriptome and are important regulators of gene expression. We identified a panel of significantly upregulated lncRNAs through analysis of a colon adenocarcinoma RNA-seq data set. These lncRNAs have been associated with tumor progression and metastasis in a number of different cancers. Our aim was to validate expression of these CRC-associated lncRNAs in fresh frozen CRC tissue and normal adjacent epithelium obtained from patients undergoing initial surgical resection.  

Methods:  CRC and normal adjacent epithelium (>10cm from CRC tissue) from eight patients were acquired from our institution’s tissue biorepository. Tissue specimens were cut, placed onto slides, and stained. Cancer and normal epithelial cells of interest were captured using the ArcturusXT™ Laser Capture Microdissection System, using H&E slides as a reference (Fig. 1A). Total RNA was extracted and expression of lncRNA (FAM83H-AS1, PVT1, UCA1, H19, FER1L4, GAS5, ZFAS1, and GAPDH as internal control) was measured using qRT-PCR. 

Results:
In CRC samples, PVT1 & ZFAS1 were significantly upregulated (FC=2.01, p=0.048 & FC= 3.17, p=0.0001, respectively) while FAM83H-AS1 & UCA1 were significantly downregulated (FC=-2.91, p=0.008 & FC=-4.85, 0.002, respectively) compared to normal adjacent epithelium (Fig. 1B). We also observed a significant difference in the expression of PVT1 (p=0.0005) and a trend toward significance in FAM83H in local (Stage I & II) versus metastatic (Stage III & IV) expression (p=0.059)(Fig. 1C). No significant difference was seen in the expression of H19, FER1L4, and GAS5 in CRC samples versus normal adjacent epithelium (Fig. 1B).

Conclusion:
Here we show the differential expression of lncRNAs that are associated with colon adenocarcinoma as compared to normal. PVT1 & ZFAS1 were shown to be significantly upregulated in CRC tissues while FAM83H-AS1 & UCA1 were significantly downregulated. PVT1 was also increased in local compared to metastatic cancers. These results validate the RNA-seq data set for the upregulated lncRNAs but are in contrast to the data set for downregulated lncRNAs. This may be due to a small study population and the use of LCM tissue versus whole tissue, which was used for the RNA-seq data set and has a broader gene expression profile. Additionally, lncRNA primers may not fully capture gene expression. Future studies will verify observed expression profiles in CRC tissues and cell lines via PVT1 & ZFAS1 knockdown and FAM83H-AS1 & UCA1 knock-in experiments.
 

45.16 Slippery Nanoparticles as a Diffusion Platform for Mucin Producing Gastrointestinal Tumors

Posted on January 31, 2019

M. Khalili1, L. Daniels1, A. Thadi1, B. Polyak1, W. F. Morano1, H. Zhou1, F. Zhiyuan1, H. Cheng1, W. B. Bowne1  1Drexel University College Of Medicine,Philadelphia, Pa, USA

Introduction:
Treatment failures in pseudomyxomaperitonei (PMP) may result from ineffective delivery of therapeutics through dense PMP mucinous tumor barriers. Nanoparticle (NP) diffusion depends on surface interaction with proteins. Thus NPs that interact weakly with mucin matrix will penetrate mucin faster.  We have modified the surface of Poly (lactic-co-glycolic acid)-b-polyethylene glycol (PEG-NPs) with a low-density, 2nd PEG layer (TPEG-NPs-20) to reduce it’sbinding affinity to proteins and improve diffusion through mucin.

Methods:
Nanoprecipitation method was used to engineer PEG-NPs. To construct the 2nd PEG layer of TPEG-NPs-20, PEG-Thiol was conjugated to PEG-NPs via maleimide-thiol reaction. DiD-labeled NPs were added to inner wells of two separate transwell diffusion systems to characterize NP diffusion through PMP tissue and cultured LS174T cells. Diffusionrate of DiD-labeled NPs through mucin was characterized by measuring fluorescence signal in the bottom well as a function of time. NP diffusion rates were recorded under shear stress (plate placed on an orbital shaker at 100 rpm) and static conditions at 37 °C. Diffusion properties of TPEG-NPs-20 were further demonstrated in ex-vivo rat small intestine mucin penetrating model. Rat small intestine was collected and submerged and in a solution of either DiD-labeled TPEG-NPs-20 or PEG-NPs for 30 mins. The intestinal loops were then washed, placed in embedding matrix, and frozen for cryosection. The sliced sections were mounted with DAPI-containing mounting media and inspected via confocal microscopy.

Results:
Diffusion profiles of TPEG-NPs-20 and PEG-NPs through PMP and cultured LS174T cells were generated. Under static conditions, there was no significant difference in the overall diffusion rate through PMP between TPEG-NPs-20 and PEG-NPs. Under shear stress, diffusion of PEG-NPs through PMP tissue remained similar to the diffusion rate at static condition. However, TPEG-NPs-20 diffused faster; after 8 hours with ~100% penetration through the PMP tissue layer vs. only ~40%  diffusion of PEG-NPs (Figure). Similarly, transwell experiment with LS174T inside the inner wells resulted in superior diffusion rate of TPEG-NPs-20 compared to PEG-NPs under shear stress conditions. Superior diffusion of TPEG-NPs-20 was also observed in ex-vivo rat small intestine mucin penetrating model. TPEG-NPs-20 showed faster diffusion under shear stress with elevated luminal NP fluorescent signals detected 30 minutes after NP addition, whereas PEG-NPs werebarely detectable on the luminal surface of the intestine.

Conclusion:
TPEG-NPs-20 can be an effective anti-cancer drug delivery system to penetrate the dense mucin barrier secreted by gastrointestinal tumors. 
 

45.15 The Combination of EZH2 and FAK Inhibition Decreases Viability of Neuroblastoma Cells in vitro

Posted on January 31, 2019

G. J. Bhatt1, A. P. Williams2, L. L. Stafman2, J. M. Aye3, J. E. Stewart2, E. A. Beierle2  3University Of Alabama at Birmingham,Hematology And Oncology,Birmingham, Alabama, USA 1Alabama College of Osteopathic Medicine,Dothan, AL, USA 2University Of Alabama at Birmingham,Surgery,Birmingham, Alabama, USA

Introduction:
Neuroblastoma is the most common pediatric extracranial solid tumor. Originating from neural crest cells, it is often unpredictable in nature. In high risk disease, the 5-year survival rate is only about 40-50%. Given its prevalence in the pediatric population and its lethality, aggressive research is needed to combat this tumor. Much promise has been found in the study of Enhancer of Zeste Homologue 2 (EZH2), a histone methyltransferase. EZH2 inhibitors are currently undergoing clinical trials for the treatment of pediatric solid tumors. EZH2 has been found to affect neuroblastoma cell survival and tumorigenesis. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase which has been proven to decrease survival and proliferation in neuroblastoma1. We have shown that there is a direct interaction between EZH2 and FAK in neuroblastoma. EZH2 inhibition, as well as FAK inhibition, have shown promise in treating many solid tumors, but combination therapy of the two has not been investigated in neuroblastoma. Our objective was to show that combined inhibition of FAK and EZH2 would decrease neuroblastoma survival and proliferation.

Methods:
Long-term passaged SH-EP and WAC2 neuroblastoma cell lines were utilized. Cells were treated with a combination of doses of GSK343 (EZH2 inhibitor) and PF-573, 228 (FAK inhibitor) on 96 well plates. Cell viability and proliferation were measured with alamarBlue® and Cell Titer96® assays, respectively. The Chou-Talalay method was used to analyze results of the combination therapy, and combination indices (CI) were calculated with a combination index less than 1 representing synergism.

Results:
The SH-EP cell line displayed synergistic effects from combination therapy of GSK343 and PF, showing markedly decreased viability after treatment. Treatment with GSK343 alone resulted in an LD50 of 63.5mM, and PF treatment alone resulted in an LD50 of 8.2mM. At a constant dose of 10mM GSK343 or 15mM GSK343, combination with 2, 4, or 6mM PF yielded combination indices of 0.875 and 0.919, respectively. Constant doses of 2 or 4mM PF combined with 5, 10, or 15mM GSK33 yielded combination indices 0.825 and 0.828, respectively. These values, which are less than one represent synergy.

Conclusion:
The SH-EP cell line displayed synergistic effects from combination therapy of GSK343 and PF, showing markedly decreased viability after treatment. In conclusion, these data indicate that further investigation of EZH2 inhibition in combination with FAK inhibition is warranted in neuroblastoma.
 

45.14 Clinical Significance of LGR5, a Cancer Stem Cell Marker in Colorectal Cancer Peritoneal Metastasis

Posted on January 31, 2019

C. U. Ihemelandu1, C. Albanese1  1Georgetown University Medical Center,Surgical Oncology,Washington, DC, USA

Introduction: Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is expressed on Wnt/β-catenin-dependent adult stem cell populations of the colon. Cancer stem cells are hypothesized to be the driving force behind tumor progression and metastasis, making them attractive therapeutic targets. Our aim was  to analyze the clinicopathologic and prognostic significance of LGR5 expression in a cohort of colorectal cancer patients with peritoneal metastasis.

Methods: A total of 49 Formalin-fixed paraffin-embedded (FFPE) tissue blocks of primary or metastatic tumors and their respective normal tissues were collected from the tissue bank for time period 2009-2015. LGR5 expression was assessed at the protein level through immunohistochemical (IHC) staining of tissue microarray (TMA) constructs consisting of pairs of tumor and normal colon tissue. The correlation between LGR5 expression and clinicopathologic  parameters and prognosis was assessed by statistical analysis.

Results:Of the 49 patient sample, 30(61.22%) were female vs. 19 (38.78%) males. Age range at initial diagnosis ranged from 31.7 years to 84.4 years, with a median age of 61.29 years. Duration of follow-up ranged from 1 – 9 years with a median of 5 years.LGR5 expression was higher in colorectal cancer than in normal mucosa. In univariate survival analysis overexpression of LGR5 was significantly associated with improved survival ( p=0.002).Of significance, LGR5 positivity was an independent prognostic marker for better prognosis in a multivariate survival analysis adjusting for prognostic variables age, stage, gender, tumor histology and grade (HR 2.67. 95% CI 1.01-7.00, P=0.046). 

Conclusion:LGR5 was significantly over expressed in colorectal cancer compared to normal tissues. LGR5 was noted to be an independent prognostic variable for an improved survival outcome in colorectal cancer patients with peritoneal metastasis, making LGR5 a potential therapeutic target in colorectal cancer patients with peritoneal metastasis.

 

45.13 Intravital imaging of the murine lung reveals the efficiency of the metastatic cascade

Posted on January 31, 2019

A. H. Coste1,2, L. Borriello1,3, Y. Wang1,3, M. Oktay1,4, J. S. Condeelis1,2,3,5, X. Ye1,3, D. Entenberg1,3,5  2Albert Einstein College of Medicine and Montefiore Medical Center,Department Of Surgery,Bronx, NY, USA 3Albert Einstein College of Medicine, Bronx,Gruss-Lipper Biophotonics Center,Bronx, NY, USA 4Department of Pathology,Albert Einstein College Of Medicine,Bronx, NY, USA 5Albert Einstein College of Medicine,Integrated Imaging Program,Bronx, NY, USA 1Albert Einstein College of Medicine and Montefiore Medical Center,Structural Anatomy And Biology,Bronx, NY, USA

Background: Breast cancer is the leading cause of cancer related-death in women[1] with over 90% of these deaths due to metastasis. In these cases, the lung is the most common anatomical site of metastasis found at autopsy[2]. For the last 100 years, the process of metastasis has been studied through the use of an experimental metastasis (EM) assay [3] consisting of tail vein injection of tumor cells into tumor-free mice, followed by histopathological analysis of the lung weeks later to gain insight into tumor cell arrival, survival, and the growth of metastases. These studies have concluded that metastasis is an inefficient process[4]. Using a new technology developed at Albert Einstein College of Medicine, called the Window for High Resolution Imaging of the Lung (WHRIL)[5], we have directly compared EM to the more clinically relevant process of tumor cells spontaneously metastasizing (SM) from a primary tumor to the lung and have found significant differences in metastatic efficiency between EM and SM.

Methods:   Real-time images of tumor cell dissemination were captured using the WHRIL (Figure 1) in both EM and SM models. Metastatic potential was compared by analyzing, and comparing between the models, the percentage of tumor cells surviving in the lung over time, their endothelial crossing-time, their frequency of interaction with macrophages, the fraction of cells which are dormant, and the percent of cells that developed into metastases.

Results: Tumor cells which spontaneously metastasize from primary tumors show a ten-fold higher rate of survival in the lung and three times greater efficiency in forming metastases compared to those directly injected into the lung vasculature. Most of SM tumor cells are dormant indicating that the residual disease phenotype is programmed by the primary tumor either directly in the primary site or indirectly at the secondary site.

Conclusion: These results indicate that experimental metastasis does not accurately reflect the true clinical process and that spontaneous dissemination from a primary tumor has significant influence on the survival and growth of disseminated cells. This suggests that the tumor microenvironment of the primary tumor educates tumor cells for survival, dormancy and growth at the primary site, and/or prepares the pre-metastatic niche, in the secondary site.  Understanding where and how disseminated tumor cells are educated is critical to preventing their survival and growth.at secondary sites. This discovery will open the door to new strategies for the treatment of metastatic tumors to prevent metastatic progression and death.

 

« Previous Page
Next Page »