Hyperoxia influences cancer growth and metastasis. A pilot experimental model

Grigore T. Popa University of Medicine and Pharmacy, Iași, România, Regional Institute of Oncology, Anaesthesia and Intensive Care Department, Iași, România, Regional Institute of Oncology, Department of Molecular Biology, TRANSCEND, Iași, România, CEMEX Research Center, Grigore T. Popa University of Medicine and Pharmacy, Iași, România, The 5th International Congress of the Society of Anesthesiology and Reanimatology of the Republic of Moldova, 16th Edition of the International Course of Guidelines and Protocols in Anesthesia, Intensive Care and Emergency Medicine, 28th Meeting of the European Society for Computing and Technology in Anesthesia and Intensive Care September 27-29, 2018, Chisinau, the Republic of MoldovaIntroduction: Perioperative care of cancer patients is under scrutiny. Among many factors promoting cancer recurrence and metastasis, high oxygen concentration exposure is underevaluated. While oxygen toxicity is documented in several circumstances, its implication in tumor cell growth and progression is poorly understood. Objective: To characterize high oxygen concentration exposure effects on tumor progression using a breast cancer murine model. Material and methods: A highly aggressive breast tumor cell line 4T1 (ATCC®) was injected in mammary gland in 8 week old females BALB/c mice. We divided the animals into 3 groups, each including 6 individuals: G1 – tumor bearing mice with no intervention post inoculation; G2 – primary tumor removal at 2 weeks post inoculation; G3 - primary tumor removal at 2 weeks post inoculation followed by 6 hours of 75% oxygen exposure. In all groups cancer evolution was assessed at 6 weeks by standard pathomorphological evaluation: specimens from the primary tumor, locally recurrent tumor and target organ metastasis were assessed by hematoxylin-eosin staining, and digital microscopy. Results: Surgically removed primary tumors in G3 group had similar characteristics with those in G2 group and previously described models. At study endpoint, compared with both G2 and G1 groups, G3 animals showed significantly higher tumor burden: larger local recurrence and more metastasis (larger number and dimensions) in liver and lungs, associated with significantly enlarged spleen. Conclusions: Short term (6 hours) high oxygen (75%) concentration exposure results in significantly more aggressive progression of a 4T1-BALB/c murine breast cancer model

Models for testing regenerative therapies – focus on explants as models for osteoarthritis

Background. Osteoarthritis (OA) is a degenerative disease that progressively involves all joint compartments leading to destruction and loss of function. Regenerative medicine (RM) aims to introduce revolutionary therapies dedicated to drastically improve the way we treat degenerative diseases including OA. Explanted cartilage tissue has been proposed as a modality to study cartilage ontogeny and to understand cartilage repair as well as modality to screen new drug/therapies for the treatment of OA. Objectives. To establish a working protocol for obtaining human osteochondral tissue explants in co -culture with synovial tissue to be used as ex vivo models for OA and to demonstrate explantreactivity to conditioned media (CM) from adipose mesenchymal cells (ADSC) tested as a modality for bone and cartilage rescue. Methods. Human osteochondral samples were collected from patients undergoing total knee replacement were kept in incomplete chondrogenic media (ICM) or in serum free DMEM.Explantreleased cytokines were quantified by. ELISA for Human tumor necrosis factor alpha(TNFα) and interleukin -6 (IL-6) and qPCR-based immunoassay for Human IL-17A and Human IL-1β (Proquantum™ immunoassay kit Invitrogen). Histology, Western blot and Immunohistochemistrystudies to detect Collagen type II (Col II) matrix metalloprotease 1 and 13 (MMP I, MMP-13),Perlecan and beta galactosidase (BGAL) are going on. Results. We found that culture media as well as synovial tissue presence influences the level ofdetectable IL-6, IL-17A CM increased IL-6 presence up to 29 days in culture; TNF α and IL1B levelsdecrease after 7 days in culture; CM treatment significantly decrease TNF α in both synoviumcontaining DMEM and ICM cultured explants Histology revealed presence of active chondrocytewith enlarged hyperchromatic nuclei in CM treated explants. Conclusion. Time in culture, type of culture media and synovial presence influence explant reactivity. Presence of synovial tissue increase explant reactivity especially for situation when an antiinflammatory effect is expected. Histology and immunohistochemistry can detect markers of tissue regeneration. Explant culture can serve as a reliable ex vivo model for testing both antiinflammatory as well as tissue remodeling intervention for articular joint repair

The Unexplored Role of Intra-articular Adipose Tissue in the Homeostasis and Pathology of Articular Joints

Intra-articular adipose tissue deposits known as articular fat pads (AFPs) are described to exist within synovial joints. Their assumed role in normal joint biomechanics is increasingly objectivized by means of advanced methods of functional imaging. AFPs possess structural similarity with body subcutaneous white adipose tissue (WAT), however, seems to be regulated by independent metabolic loops. AFP dimension are conserved during extreme WAT states: obesity, metabolic syndrome, lipodystrophy, and cachexia. Hoffa fat pad (HFP) in the knee is increasingly recognized as a major player in pathological joint states such as anterior knee pain and osteoarthritis. HFP contains numerous population of mesenchymal and endothelial progenitors; however, the possible role of mature adipocytes in the maintenance of stem cell niche is unknown. We propose that AFP is an active component of the joint organ with multifunctional roles in the maintenance of joint homeostasis. Endowed with a rich network of sensitive nervous fibbers, AFPs may act as a proprioceptive organ. Adipokines and growth factors released by AFP-resident mature adipocytes could participate in the maintenance of progenitor stem cell niche as well as in local immune regulation. AFP metabolism may be locally controlled, correlated with but independent of WAT homeostasis. The identification of AFP role in normal joint turnover and its possible implication in pathological states could deliver diagnostic and therapeutic targets. Drug and/or cell therapies that restore AFP structure and function could become the next step in the design of disease modifying therapies for disabling joint conditions such as osteoarthritis and inflammatory arthritis

Isolation and Phenotypic Characterisation of Stem Cells from Late Stage Osteoarthritic Mesenchymal Tissues

Introduction: Osteoarthritis (OA) represents an increasing health issue worldwide. Regenerative medicine (RM) has raised the hope for introducing revolutionary therapies in clinical practice. Detection of autologus cell sources can improve accessibility to RM strategies. Objectives: To assess the presence and biological potential of mesehchymal stem cells in three tissues (subchondral bone, synovial layer, periarticular adipose tissue) in late stages osteoarthritic patients. Material and Methods: Samples were collected from subjects undergoing total knee replacement (TKR). MSCs were isolated and cultured in complete αMEM with β FGF. Cell morphology and growth potential was assessed. Flow cytometry was used for detection of several relevant cell surface markers. Quantitative and qualitative assessment of differentiation potential towards three mesenchymal lineages (osteogenesis adipogenesis chondrogenesis) was performed. Time lapse life cell imaging of nondiferentiated cells over 24 hours period was used to determine cell kinetics. Results: Mesenchymal cells derived from all donors and tissue types showed morphology, growth and surface cell markers associated with stemness. All cell types underwent differentiation toward three mesenchymal lineages with significant differences between tissues of origin, not between donors. Cell kinetics, as derived from life imaging records, was variable with tissue of origin, significant higher for adipose derived MSCS. Conclusion: Human late stage OA mesenchymal tissues, contain progenitors with proliferative and differentiation potential of MSCs. These populations can be used for research and autologus regenerative therapies. Further comparative studies with age matched non OA samples has the potential of contributing to deepening knowledge about disease occurrence and progression.Deposited by bulk impor

Isolation and phenotypic characterisation of stem cells from late stage osteoarthritic mesenchymal tissues

Introduction: Osteoarthritis (OA) represents an increasing health issue worldwide. Regenerative medicine (RM) has raised the hope for introducing revolutionary therapies in clinical practice. Detection of autologus cell sources can improve accessibility to RM strategies. Objectives: To assess the presence and biological potential of mesehchymal stem cells in three tissues (subchondral bone, synovial layer, periarticular adipose tissue) in late stages osteoarthritic patients. Material and Methods: Samples were collected from subjects undergoing total knee replacement (TKR). MSCs were isolated and cultured in complete alpha MEM with beta FGF. Cell morphology and growth potential was assessed. Flow cytometry was used for detection of several relevant cell surface markers. Quantitative and qualitative assessment of differentiation potential towards three mesenchymal lineages (osteogenesis adipogenesis chondrogenesis) was performed. Time lapse life cell imaging of nondiferentiated cells over 24 hours period was used to determine cell kinetics. Results: Mesenchymal cells derived from all donors and tissue types showed morphology, growth and surface cell markers associated with stemness. All cell types underwent differentiation toward three mesenchymal lineages with significant differences between tissues of origin, not between donors. Cell kinetics, as derived from life imaging records, was variable with tissue of origin, significant higher for adipose derived MSCS. Conclusion: Human late stage OA mesenchymal tissues, contain progenitors with proliferative and differentiation potential of MSCs. These populations can be used for research and autologus regenerative therapies. Further comparative studies with age matched non OA samples has the potential of contributing to deepening knowledge about disease occurrence and progression

Long-Term Deleterious Effects of Short-term Hyperoxia on Cancer Progression—Is Brain-Derived Neurotrophic Factor an Important Mediator? An Experimental Study

Perioperative factors promoting cancer recurrence and metastasis are under scrutiny. While oxygen toxicity is documented in several acute circumstances, its implication in tumor evolution is poorly understood. We investigated hyperoxia long-term effects on cancer progression and some underlying mechanisms using both in vitro and in vivo models of triple negative breast cancer (TNBC). We hypothesized that high oxygen exposure, even of short duration, may have long-term effects on cancer growth. Considering that hyperoxic exposure results in reactive oxygen species (ROS) formation, increased oxidative stress and increased Brain-Derived Neurotrophic Factor (BDNF) expression, BDNF may mediate hyperoxia effects offering cancer cells a survival advantage by increased angiogenesis and epithelial mesenchymal transition (EMT). Human breast epithelial MCF10A, human MDA-MB-231 and murine 4T1 TNBC were investigated in 2D in vitro system. Cells were exposed to normoxia or hyperoxia (40%, 60%, 80% O2) for 6 h. We evaluated ROS levels, cell viability and the expression of BDNF, HIF-1α, VEGF-R2, Vimentin and E-Cadherin by immunofluorescence. The in vivo model consisted of 4T1 inoculation in Balb/c mice and tumor resection 2 weeks after and 6 h exposure to normoxia or hyperoxia (40%, 80% O2). We measured lung metastases and the same molecular markers, immediately and 4 weeks after surgery. The in vitro study showed that short-term hyperoxia exposure (80% O2) of TNBC cells increases ROS, increases BDNF expression and that promotes EMT and angiogenesis. The in vivo data indicates that perioperative hyperoxia enhances metastatic disease and this effect could be BDNF mediated

Immunological Study Regarding the Role of Ca(OH)2 Paste on the MMP8 Expression for Teeth with Chronic Periapical Lesions

Introduction MMP8 are secreted as inactive proproteins, stored in secondary granules within neutrophils and are activated by autolytic cleavage. The function of MMP8 is degradation of type I, II and III collagens. In this context, the concentration of MMP8 can be related to the intensity of inflammatory processes associated with chronic periapical lesions. Objectives The aim of our study was to measure changing of MMP8 within the periapical secretion of teeth affected by periapical lesions and treated with antibacterial medication, using immunological tests. Methods Study group included 22 patients with age 22-64 years. A number of 30 teeth with periapical lesions (periapical granuloma and diffuse periapical osteitis) were submitted to endodontic treatment and filled with Ca(OH)2 paste. The periapical secretion was collected with paper points at baseline, after 14 days and after 28 days. The concentration of MMP8 was assessed using ELISA test Quantikinine (Human MMP-8 Immunoassay, R&D System, USA) based on quantitative sandwich enzyme immunoassay. Results and discussions At baseline the mean concentration of MMP8 was 31.3 ng/mL. The concentrations of MMP8 were closely related to the type of periapical lesion: 12.5 ng/mL for incipient periapical lesions, 18.1 ng/mL for small periapical granuloma and 91.6 ng/mL for extended periapical granuloma. The levels of MMP8 decreased gradually after 2 weeks and 4 weeks comparing with baseline. Conclusion Metaloproteinases (MMP8) could be used as biochemical markers of the periapical status of inflammatory processes in course of initial stage of endodontic therapy

Optimisation of the quantitative analysis of inflammatory cell infiltrates in breast cancer /Optimizarea analizei cantitative a infiltratului celular inflamator în cancerul mamar

În acest studiu am analizat modalitatea optimă de poziţionare a cut off-ului în cadrul analizei cantitative a infiltratului inflamator în cancerul mamar cu ajutorul sistemului HistoQuest. Am utilizat specimene de ţesut mamar tumoral, care au fost marcate imunohistochimic cu markerii CD68 (pentru macrofage) şi CD8 (pentru limfocitul T citotoxic) şi ulterior analizate automat pe regiunile IT (intratumoral), PT (peritumoral) şi FI (front de invazie) cu ajutorul sistemului soft HistoQuest. Pentru evidenţierea populaţiei de celule pozitive pe histograme şi scatergrame, este necesară poziţionarea cut off-ului (discriminator). Pentru optimizarea tehnicii, am comparat 5 modalităţi de poziţionare a cut off-ului. Rezultatele obţinute statistic pentru markerul CD8 pentru toate cele 5 tipuri de cutoff aplicate pe regiunile IT, PT şi FI nu au înregistrat diferenţe semnificative statistic (p < 0,05). Pentru markerul CD68 s-au înregistrat diferenţe semnificative statistic (p<0,05) între cut off-urile alese pe criterii manuale (cut off-ul C2-manual şi cut off -ul C3-medie manual) versus cut off-urile stabilite pe criterii ce ţin de soft (cut off-ul C1-automat, cut off-ul C4-regiune negativă şi cut off-ul C5-medie automat), utilizarea cutoff-ului automat fiind preferat pentru a îndepărta factorul subiectiv. Utilizarea modalităţii de poziţionare automată a cut off-ului generează date obiective şi reproductibile şi poate fi utilizat în analize cantitative ulterioar

Mesenchymal Stem Cell-Derived Exosomes Modulate Angiogenesis in Gastric Cancer

Individualized gastric cancer (GC) treatment aims at providing targeted therapies that translate the latest research into improved management strategies. Extracellular vesicle microRNAs have been proposed as biomarkers for GC prognosis. Helicobacter pylori infection influences the therapeutic response to and the drivers of malignant changes in chronic gastritis. The successful use of transplanted mesenchymal stem cells (MSCs) for gastric ulcer healing has raised interest in studying their effects on tumor neovascularization and in potential antiangiogenic therapies that could use mesenchymal stem cell secretion into extracellular vesicles—such as exosomes—in GC cells. The use of MSCs isolated from bone marrow in order to achieve angiogenic modulation in the tumor microenvironment could exploit the inherent migration of MSCs into GC tissues. Bone marrow-derived MSCs naturally present in the stomach have been reported to carry a malignancy risk, but their effect in GC is still being researched. The pro- and antiangiogenic effects of MSCs derived from various sources complement their role in immune regulation and tissue regeneration and provide further understanding into the heterogeneous biology of GC, the aberrant morphology of tumor vasculature and the mechanisms of resistance to antiangiogenic drugs