Background: Despite many efforts to effectively treat PDAC, PDAC carries one of the highest mortality rates of all major cancers. Thus, there is a critical unmet need to develop novel approaches to improve the clinical outcome of PDAC. It is well known that many cancers, including PDAC, generate a local TME that allows cancer to escape normal immune surveillance. Phosphatidylserine (PS), a negatively charged phospholipid that is abundant on the cancer cell membrane and with known actions to promote the secretion of immunomodulatory proteins, may provide a mechanism to regulate the TME. This study explored that possibility. Methods: MΦ differentiation and polarization were assessed by Western blotting and flow cytometric approaches. PS exposure and surface markers were analyzed by flow cytometry. Protein–protein and protein–lipid interactions were analyzed by immunofluorescence and enzyme-linked immunosorbent assay (ELISA). Phospholipid and SapC-DOPG treatment were employed to assess target protein functions in MΦ polarization, tumor growth, and survival in subcutaneous and orthotopic tumor models. The PK-PD and safety of SapC-DOPG were tested on orthotopic mouse models. Results: Our studies show that PDAC secretes Hsp70 that stimulates the MΦ polarization to the immunosuppressive M2 phenotype. We found that high surface PS on cancer cells correlates with increased secretion of Hsp70 and is associated with higher MΦ differentiation activity in vitro and in vivo. Furthermore, blocking cancer cell-secreted Hsp70 with SapC-DOPG reverses the immune suppression and reduces tumor growth. Conclusions: These preclinical results reveal a novel immunotherapeutic approach to potentially improve the outcome of PDAC treatment in humans.

Glioblastoma (GBM) is an aggressive brain tumor associated with high post-therapy recurrence and very poor survival rates. One of the factors contributing to the aggressive nature of this disease is the level of heterogeneity seen at the phenotypic and genetic level. Glioma stem cells (GSCs) are stem-like cells within the tumor with the ability to self-renew and give rise to different types of cells within the tumor, hence giving rise to the heterogeneity found in GBM. GSCs are often implicated in the resistance of glioma to standard of care radiation and chemotherapy. The physical niche within a tumor mass supports stemness and aggressive characteristics of GSCs, hence, experimental systems providing a relevant tumor microenvironment (TME) are critical for adequate assessment of molecular mechanisms regulating GSC populations. Although mouse models continue to be an integral part of an in vivo experimental design, they are neither time- nor cost-efficient. Danio rerio (zebrafish) patient-derived xenografts (PDXs) overcome several of the obstacles of the mammalian systems. Zebrafish constitute a robust, easily reproducible experimental model allowing for relevant investigation of GSC populations with TME. This chapter describes methods required for generation of zebrafish PDXs to study aspects of GSC-mediated tumorigenesis and interactions with the TME.

Supplementary Data from Cancer-Selective Targeting and Cytotoxicity by Liposomal-Coupled Lysosomal Saposin C Protein

Significance Glutamate-ureido-lysine (GUL) probes are specific for prostate-specific membrane antigen (PSMA), overexpressed by most prostate cancers. This antigen can be lost as the cancer progresses. Recent reports have indicated that GUL probes can still identify these PSMA-negative tumors, indicating that the expression of alternative PSMA-like proteins may change during disease progression. In this study we identified two such candidate protein targets, NAALADaseL and mGluR8, by using a combined computational chemistry, data mining, molecular biology, radiochemistry, and synthetic chemistry approach. This work consequently prepares the groundwork for developing specific probes that can identify this progression, indicates directions for neuroendocrine prostate cancer research, and highlights the utility of a multidisciplinary approach for the rapid identification of unidentified proteins interacting with diagnostic probes.

Pediatric sepsis is a leading cause of morbidity and mortality in children. One of the most common and devastating morbidities is sepsis-related acute kidney injury (AKI). AKI was traditionally thought to be related to low perfusion and acute tubular necrosis. However, little acute tubular necrosis can be found following septic AKI, and little is known about the mechanism of septic AKI. Olfactomedin-4 (OLFM4) is a secreted glycoprotein that marks a subset of neutrophils. Increased expression of OLFM4 in the blood is associated with worse outcomes in sepsis. Here, we investigated a pediatric model of murine sepsis using murine pups to investigate the mechanisms of OLFM4 in sepsis. When sepsis was induced in murine pups, survival was significantly increased in OLFM4-null pups. Immunohistochemistry at 24 h after the induction of sepsis demonstrated increased expression of OLFM4 in the kidney, which was localized to the loop of Henle. Renal cell apoptosis and plasma creatinine were significantly increased in wild-type versus OLFM4-null pups. Finally, bone marrow transplant suggested that increased OLFM4 in the kidney reflects local production rather than filtered from the plasma. These results demonstrate renal expression of OLFM4 for the first time and suggest that a kidney-specific mechanism may contribute to survival differences in OLFM4-null animals.

An in vivo study was conducted using a mouse tumor model, to assess the utility of using gold nanoparticles (gNPs) during HIFU procedures to locally enhance heating at low powers. Tumors were grown using melanoma tumor cells (B16/F10) subcutaneously on the right flanks of mice (C57Bl/6J). Physiologically relevant concentrations (0 and 0.125%) of gNPs were directly injected into the tumors. Sonications at acoustic powers of 10 and 30 W were performed for a duration of 16 s inside a magnetic-resonance system. Temperature increases and lesion volumes were calculated and compared for procedures with and without gNPs. Histopathology study was conducted using a cleaved caspase 3 antibody and hematoxylin and eosin staining after removing the tumors from the mice. For an acoustic power of 30 W, end-of-sonication temperature increases of 25.4 ± 3.8 °C (0% gNP) and 42.2 ± 4.6 °C (0.125% gNP) were measured. Using cleaved caspase 3 antibody, it was observed that more than 1% of nuclei are affected in the case of 0.125% and 30 W but only 0.01% of nuclei are affected in the 0% case. For 30 W and a gNP concentration of 0.125%, a lesion volume of 0.33 ± 0.22 mm3 was obtained, while no lesion was observed without gNP's.

Although the incidence of de novo neuroendocrine prostate cancer (PC) is rare, recent data suggest that low expression of prostate-specific membrane antigen (PSMA) is associated with a spectrum of neuroendocrine hallmarks and androgen receptor (AR) suppression in PC. Previous clinical reports indicate that PCs with a phenotype similar to neuroendocrine tumors can be more amenable to imaging by 18F-FDG than by PSMA-targeting radioligands. In this study, we evaluated the association between neuroendocrine gene signature and 18F-FDG uptake–associated genes including glucose transporters (GLUTs) and hexokinases, with the goal of providing a genomic signature to explain the reported 18F-FDG avidity of PSMA-suppressed tumors. Methods: Data-mining approaches, cell lines, and patient-derived xenograft models were used to study the levels of 14 members of the SLC2A family (encoding GLUT proteins), 4 members of the hexokinase family (genes HK1–HK3 and GCK), and PSMA (FOLH1 gene) after AR inhibition and in correlation with neuroendocrine hallmarks. Also, we characterize a neuroendocrine-like PC (NELPC) subset among a cohort of primary and metastatic PC samples with no neuroendocrine histopathology. We measured glucose uptake in a neuroendocrine-induced in vitro model and a zebrafish model by nonradioactive imaging of glucose uptake using a fluorescent glucose bioprobe, GB2-Cy3. Results: This work demonstrated that a neuroendocrine gene signature associates with differential expression of genes encoding GLUT and hexokinase proteins. In NELPC, elevated expression of GCK (encoding glucokinase protein) and decreased expression of SLC2A12 correlated with earlier biochemical recurrence. In tumors treated with AR inhibitors, high expression of GCK and low expression of SLC2A12 correlated with neuroendocrine histopathology and PSMA gene suppression. GLUT12 suppression and upregulation of glucokinase were observed in neuroendocrine-induced PC cell lines and patient-derived xenograft models. A higher glucose uptake was confirmed in low-PSMA tumors using a GB2-Cy3 probe in a zebrafish model. Conclusion: A neuroendocrine gene signature in neuroendocrine PC and NELPC associates with a distinct transcriptional profile of GLUTs and hexokinases. PSMA suppression correlates with GLUT12 suppression and glucokinase upregulation. Alteration of 18F-FDG uptake–associated genes correlated positively with higher glucose uptake in AR- and PSMA-suppressed tumors. Zebrafish xenograft tumor models are an accurate and efficient preclinical method for monitoring nonradioactive glucose uptake.

Prostate-specific membrane antigen (PSMA) is overexpressed in most prostate adenocarcinoma (AdPC) cells and acts as a target for molecular imaging. However, some case reports indicate that PSMA-targeted imaging could be ineffectual for delineation of neuroendocrine (NE) prostate cancer (NEPC) lesions due to the suppression of the PSMA gene ( FOLH1 ). These same reports suggest that targeting somatostatin receptor type 2 (SSTR2) could be an alternative diagnostic target for NEPC patients. This study evaluates the correlation between expression of FOLH1 , NEPC marker genes and SSTR2 . We evaluated the transcript abundance for FOLH1 and SSTR2 genes as well as NE markers across 909 tumors. A significant suppression of FOLH1 in NEPC patient samples and AdPC samples with high expression of NE marker genes was observed. We also investigated protein alterations of PSMA and SSTR2 in an NE-induced cell line derived by hormone depletion and lineage plasticity by loss of p53. PSMA is suppressed following NE induction and cellular plasticity in p53-deficient NEPC model. The PSMA-suppressed cells have more colony formation ability and resistance to enzalutamide treatment. Conversely, SSTR2 was only elevated following hormone depletion. In 18 NEPC patient-derived xenograft (PDX) models we find a significant suppression of FOLH1 and amplification of SSTR2 expression. Due to the observed FOLH1 -supressed signature of NEPC, this study cautions on the reliability of using PMSA as a target for molecular imaging of NEPC. The observed elevation of SSTR2 in NEPC supports the possible ability of SSTR2-targeted imaging for follow-up imaging of low PSMA patients and monitoring for NEPC development.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. A variety of factors can contribute to the onset of this disease, including viral infection, obesity, alcohol abuse and non-alcoholic fatty liver disease (NAFLD). These stressors predominantly introduce chronic inflammation leading to liver cirrhosis and finally the onset of HCC; however, approximately 20% of HCC cases arise in the absence of cirrhosis via a poorly defined mechanism. The atypical cyclin-like protein Spy1 is capable of overriding cell cycle checkpoints, promoting proliferation and has been implicated in HCC. We hypothesize that Spy1 promotes sustained proliferation making the liver more susceptible to accumulation of deleterious mutations, leading to the development of non-cirrhotic HCC. We report for the first time that elevation of Spy1 within the liver of a transgenic mouse model leads to enhanced spontaneous liver tumourigenesis. We show that the abundance of Spy1 enhanced fat deposition within the liver and decreased the inflammatory response. Interestingly, Spy1 transgenic mice have a significant reduction in fibrosis and sustained rates of hepatocyte proliferation, and endogenous levels of Spy1 are downregulated during the normal fibrotic response. Our results provide support that abnormal regulation of Spy1 protein drives liver tumorigenesis in the absence of elevated fibrosis and, hence, may represent a potential mechanism behind non-cirrhotic HCC. This work may implicate Spy1 as a prognostic indicator and/or potential target in the treatment of diseases of the liver, such as HCC. The cyclin-like protein Spy1 enhances lipid deposition and reduces fibrosis in the liver. Spy1 also promotes increased hepatocyte proliferation and onset of non-cirrhotic hepatocellular carcinoma (HCC). Thus, Spy1 may be used as a potential target in the treatment of HCC.

BACKGROUND:Guided growth has long been used in the lower extremities but has not been applied to varus or valgus deformity in the hip, as may occur in children with cerebral palsy or developmental dysplasia of the hip. The purpose of this study was to determine if screw, plate, or drilling techniques decreased the femoral neck-shaft angle (NSA) and articular trochanteric disease (ATD), as well as describe growth plate structural changes with each method.METHODS:Twelve 8-week-old lambs underwent proximal femoral hemiepiphysiodesis (IACUC approved) using either a screw (n=4), plate (n=4), or drilling procedure (n=4). Postoperative time was 6 months. Radiographs taken after limb harvest were used to measure NSA and ATD. Differences between treated and control sides were determined by 1-tailed paired t tests and Bonferroni (α=0.05/3). Histology was obtained for 1 limb pair per group. Proximal femurs were cut in midcoronal plane and the longitudinal growth plates were examined for structural changes.RESULTS:The mean NSA measured 7 degrees less than controls in this model using the screw technique, and this difference was statistically significant. Differences between the control and the treated groups did not reach statistical significance for either the plate or the drill group. Differences in ATD were not statistically significant, although there was a trend for larger ATD measurements using the screw technique. Histologically, physeal changes were observed on the operative sides in screw and plate specimens, but not drill specimens, compared with contralateral sham control. The screw specimen exhibited the most severe changes, with growth plate closure over half the section. The plate specimen showed focal loss of the physis across the section, but with no evidence of closure.CONCLUSIONS:This study builds on previous work that indicates screw hemiepiphysiodesis can effectively alter the shape of the proximal femur, and result in a lower neck-shaft ankle (or lesser valgus). This study suggests that implantation of a screw is likely to be more effective than a plate or drilling procedure in decreasing the NSA in skeletally immature hips.CLINICAL SIGNIFICANCE:If further preclinical, and later clinical, studies demonstrate reproducible efficacy, guided growth of the proximal femur may eventually become a viable option for treatment or prevention of hip deformity in select patients.

Severe sepsis and septic shock are the biggest cause of mortality in critically ill patients. Obesity today is one of the world's greatest health challenges. Little is known about the extent of involvement of the white adipose tissue (WAT) in sepsis and how it is being modified by obesity. We sought to explore the involvement of the WAT in sepsis. We hypothesize that sepsis induces browning of the WAT and that obesity alters the response of WAT to sepsis. Six-week-old C57BL/6 mice were randomized to a high-fat diet to induce obesity (obese group) or control diet (nonobese group). After 6 to 11 weeks of feeding, polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Mice were sacrificed at 0, 18, and 72 h after CLP and epididymal WAT (eWAT), inguinal WAT, and brown adipose tissue (BAT) harvested. Both types of WAT were processed for light microscopy and transmission electron microscopy to assess for morphological changes in both obese and nonobese mice. Tissues were processed for immunohistochemistry, image analyses, and molecular analyses. BATs were used as a positive control. Nonobese mice have an extensive breakdown of the unilocular lipid droplet and smaller adipocytes in WAT compared with obese mice after sepsis. Neutrophil infiltration increases in eWAT in nonobese mice after sepsis but not in obese mice. Nonobese septic mice have an increase in mitochondrial density compared with obese septic mice. Furthermore, nonobese septic mice have an increase in uncoupling protein-1 expression. Although the WAT of nonobese mice have multiple changes characteristic of browning during sepsis, these changes are markedly blunted in obesity.

Background: The origin of juvenile osteochondritis dissecans (OCD) is unknown. Existing experimental animal models of OCD most frequently involve surgically created lesions but do not examine repetitive stress as a possible cause of OCD. Hypothesis: Repetitive stresses can cause OCD-like lesions in immature animals. Study Design: Controlled laboratory study. Methods: Six juvenile rabbits were subjected to repetitive loading forces of approximately 160% body weight to the right hindlimb during five 45-minute sessions per week for 5 weeks. The contralateral limb was the unloaded control. After 5 weeks, rabbits were euthanized and examined with radiographs, micro–computed tomography, and gross and histopathologic analysis. Results: All 6 rabbits developed osteochondral lesions in loaded limbs on the medial and lateral femoral condyles, while contralateral unloaded limbs did not demonstrate lesions. Loaded limbs developed relative osteopenia in the femoral epiphysis and tibial metaphysis with associated decreased trabecular density. Loaded limbs also demonstrated increased femoral subchondral bone thickness near the lesions. Lesions did not have grossly apparent extensive articular cartilage damage; however, cartilage thickness increased on histology with reduced ossification. Loaded knees demonstrated abundant chondrocyte cloning, limited cartilage fissuring, and a focal loss of cellularity at the articular surface. Conclusion: Low-grade lesions in human OCD have little gross articular cartilage involvement despite substantial changes to the subchondral bone as shown on magnetic resonance imaging and radiographs. Histopathology findings in this study included cartilage thickening and chondrocyte cloning resembling those of recently published human OCD biopsy studies. Our animal model supports the hypothesis that repetitive stress to immature knees may contribute to the development of human OCD. This model may be useful in understanding the pathophysiology and healing of human OCD. Clinical Relevance: Repetitive physiologic stress generated changes to the subchondral bone in immature animals without causing extensive articular damage. The similarities of these lesions in gross and histologic appearance with human OCD support repetitive stress as the likely the cause for human OCD.

OBJECTIVE. The objective of our study was to correlate specimens of juvenile osteochondritis dissecans (OCD) lesions of the knee to MRI examinations to elucidate the histopathologic basis of characteristic imaging features.MATERIALS AND METHODS. Five children (three boys and two girls; age range, 12-13 years old) who underwent transarticular biopsy of juvenile OCD lesions of the knee were retrospectively included in this study. Two radiologists reviewed the MRI examinations and a pathologist reviewed the histopathologic specimens and recorded characteristic features. Digital specimen photographs were calibrated to the size of the respective MR image with the use of a reference scale. Photographs were rendered semitransparent and overlaid onto the MR image with the location chosen on the basis of the site of the prior biopsy.RESULTS. A total of seven biopsy specimens were included. On MRI, all lesions showed cystlike foci in the subchondral bone, bone marrow edema pattern on proton density-or T2-weighted images, and relatively thick unossified epiphyseal cartilage. In four patients, a laminar signal intensity pattern was seen, and two patients had multiple breaks in the subchondral bone plate. Fibrovascular tissue was found at histopathology in all patients. Cleft spaces near the cartilage-bone interface and were seen in all patients while chondrocyte cloning was present in most cases. Focal bone necrosis and inflammation were infrequent MRI findings. Precise correlation of the MRI appearance to the histopathologic overlays consistently was found.CONCLUSION. A direct correlation exists between the histopathologic findings and the MRI features in patients with juvenile OCD. Additional studies are needed to correlate these MRI features with juvenile OCD healing success rates.

AJR:205, July 2015 and of which patient is likely to respond is important because selection of the appropriate therapy will allow timely symptomatic relief and a return to normal activities. If a juvenile OCD lesion is left untreated or unhealed, there is a potential for early onset of degenerative joint disease. A previous study that evaluated the histopathology of juvenile OCD lesions indicated that deep instability at the OCD site was possible even if lesions were stable at arthroscopy [7]. This finding possibly accounts for the subset of patients with stable-appearing OCD lesions that do not respond to nonoperative management and suggests that early operative intervention may be better suited for some children [7]. Therefore, the identification of advanced imaging correlates to histopathologic findings is important to triage patients to the appropriate treatment. However, to date, there is no precise correlation of histopathologic findings to the MRI appearance of a juJuvenile Osteochondritis Dissecans: Correlation Between Histopathology and MRI

Through a recently conducted rat-tail vibration experiment, we have been able to determine that the tested frequencies of vibration have a significant effect on biochemical damage signified by nitro-tyrosine (NT) staining on trabecular bone, while structural damage quantified through a Hematoxylin and Eosin (H&E) stain on cortical bone exhibited statistical significance only for the 250 Hz group compared to the control group. These results seem to indicate a relationship between the growing quantities of biochemical damage when increasing the excitation frequency, thus further experimentation for frequencies between 250 Hz and 400 Hz is recommended.

Nature 517, 501–504 (2015); doi:10.1038/nature13868 Owing to a production error, in Fig. 1b of this Letter, the key should have shown that the black bars corresponded to ‘WT’ and the red bars to ‘γ1−’, instead of the other way round. In addition, in Fig. 1c the ‘WT’ label was missing from the mouse on the left, and the ‘γ1−’ label should have applied to the mouse on the right.

Hand-Arm Vibration Syndrome (HAVS) is caused by hand-transmitted vibration in industrial workers. Current ISO guidelines (ISO 5349) might underestimate vascular injury associated with range of vibration frequencies near resonance. A rat-tail model was used to investigate the effects of higher frequencies >100 Hz on early vascular damage. 13 Male Sprague-Dawley rats (250 ± 15 gm) were used. Rat-tails were vibrated at 125 Hz and 250 Hz (49 m/s(2)) for 1D, 5D and 10D; D=days (4 h/day). Structural damage of the ventral artery was quantified by vacuole count using Toluidine blue staining whereas biochemical changes were assessed by nitrotyrosine (NT) staining. The results were analyzed using one-way repeated measures mixed-model ANOVA at p<0.05 level of significance. The structural damage increased at 125 Hz causing significant number of vacuoles (40.62 ± 9.8) compared to control group (8.36 ± 2.49) and reduced at 250 Hz (12.33 ± 2.98) compared to control group (8.36 ± 2.49). However, the biochemical alterations (NT-signal) increased significantly for 125 Hz (143.35 ± 5.8 gray scale value, GSV) and for 250 Hz (155.8 ± 7.35 GSV) compared to the control group (101.7 ± 4.18 GSV). Our results demonstrate that vascular damage in the form of structural and bio chemical disruption is significant at 125 Hz and 250 Hz. Hence the current ISO guidelines might underestimate vascular damage at frequencies>100 Hz.

Whenever a tendon or its bone insertion is disrupted or removed, existing surgical techniques provide a temporary connection or scaffolding to promote healing, but the interface of living to non-living materials soon breaks down under the stress of these applications, if it must bear the load more than acutely. Patients are thus disabled whose prostheses, defect size, or mere anatomy limit the availability or outcomes of such treatments. Our group developed the OrthoCoupler™ device to join skeletal muscle to prosthetic or natural structures without this interface breakdown. In this study, the goat knee extensor mechanism (quadriceps tendon, patella, and patellar tendon) was removed from the right hind limb in 16 goats. The device connected the quadriceps muscle to a stainless steel bone plate on the tibia. Mechanical testing and histology specimens were collected from each operated leg and contralateral unoperated control legs at 180 days. Maximum forces in the operated leg (vs. unoperated) were 1,400 ± 93 N (vs. 1,179 ± 61 N), linear stiffnesses were 33 ± 3 N/mm (vs. 37 ± 4 N/mm), and elongations at failure were 92.1 ± 5.3 mm (vs. 68.4 ± 3.8 mm; mean ± SEM). Higher maximum forces (p = 0.02) and elongations at failure (p=0.008) of legs with the device versus unoperated controls were significant; linear stiffnesses were not (p=0.3). We believe this technology will yield improved procedures for clinical challenges in orthopedic oncology, revision arthroplasty, tendon transfer, and tendon injury reconstruction.