Circulating tumour cells from patients with colorectal cancer have cancer stem cell hallmarks in ex vivo culture.

OBJECTIVE: Although counting of circulating tumour cells (CTC) has attracted a broad interest as potential markers of tumour progression and treatment response, the lack of functional characterisation of these cells had become a bottleneck in taking these observations to the clinic. Our objective was to culture these cells in order to understand them and exploit their therapeutic potential to the full. DESIGN: Here, hypothesising that some CTC potentially have cancer stem cell (CSC) phenotype, we generated several CTC lines from the blood of patients with advanced metastatic colorectal cancer (CRC) based on their self-renewal abilities. Multiple standard tests were then employed to characterise these cells. RESULTS: Our CTC lines self-renew, express CSC markers and have multilineage differentiation ability, both in vitro and in vivo. Patient-derived CTC lines are tumorigenic in subcutaneous xenografts and are also able to colonise the liver after intrasplenic injection. RNA sequencing analyses strikingly demonstrate that drug metabolising pathways represent the most upregulated feature among CTC lines in comparison with primary CRC cells grown under similar conditions. This result is corroborated by the high resistance of the CTC lines to conventional cytotoxic compounds. CONCLUSIONS: Taken together, our results directly demonstrate the existence of patient-derived colorectal CTCs that bear all the functional attributes of CSCs. The CTC culture model described here is simple and takes <1 month from blood collection to drug testing, therefore, routine clinical application could facilitate access to personalised medicine. CLINICAL TRIAL REGISTRATION: ClinicalTrial.gov NCT01577511.

GPR40, a free fatty acid receptor, differentially impacts osteoblast behavior depending on differentiation stage and environment.

GPR40 is a free fatty acid receptor that has been recently shown to impact bone remodeling. This receptor protects skeleton by inhibiting bone resorbing osteoclast differentiation. Consistent with GPR40 expression on bone forming cells, we assumed that this receptor may also influence osteoblast activity. To further investigate this hypothesis, biological effects of GW9508, a synthetic agonist for GPR40, was first tested on osteoblast differentiation parameters. Assays were performed in two different cell models: the MC3T3-E1 osteoblastic cell line and primary bone marrow cultures extracted from wild-type and GPR40 knock-out mice. Both models showed a dual role of GPR40 on osteoblasts. Although receptor stimulation induced early stimulation of differentiation marker expression, it finally led to inhibition of mineralization process during late differentiation stages. To further elucidate this discrepancy, mice were ovariectomized to induce bone loss and received GPR40 agonist by gavage. Data revealed a weak influence of GPR40 agonist on osteoblast markers expression. Nevertheless, a significant increase in OPG expression was observed upon GW9508 treatment that contribute to explain the GPR40-related osteoporosis prevention. To conclude, our results confirm the relevance of this new opportunity in the management of bone loss.

The free fatty acid receptor G protein-coupled receptor 40 (GPR40) protects from bone loss through inhibition of osteoclast differentiation.

The mechanisms linking fat intake to bone loss remain unclear. By demonstrating the expression of the free fatty acid receptor G-coupled protein receptor 40 (GPR40) in bone cells, we hypothesized that this receptor may play a role in mediating the effects of fatty acids on bone remodeling. Using micro-CT analysis, we showed that GPR40(-/-) mice exhibit osteoporotic features suggesting a positive role of GPR40 on bone density. In primary cultures of bone marrow, we showed that GW9508, a GRP40 agonist, abolished bone-resorbing cell differentiation. This alteration of the receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation occurred via the inhibition of the nuclear factor κB (NF-κB) signaling pathway as demonstrated by decrease in gene reporter activity, inhibitor of κB kinase (IKKα/ß) activation, inhibitor of κB (IkBα) phosphorylation, and nuclear factor of activated T cells 1 (NFATc1) expression. The GPR40-dependent effect of GW9508 was confirmed using shRNA interference in osteoclast precursors and GPR40(-/-) primary cell cultures. In addition, in vivo administration of GW9508 counteracted ovariectomy-induced bone loss in wild-type but not GPR40(-/-) mice, enlightening the obligatory role of the GPR40 receptor. Then, in a context of growing prevalence of metabolic and age-related bone disorders, our results demonstrate for the first time in translational approaches that GPR40 is a relevant target for the design of new nutritional and therapeutic strategies to counter bone complications.

GW9508, a free fatty acid receptor agonist, specifically induces cell death in bone resorbing precursor cells through increased oxidative stress from mitochondrial origin.

GW9508 is a free fatty acid receptor agonist able to protect from ovariectomy-induced bone loss in vivo thought inhibition of osteoclast differentiation in a G-coupled Protein Receptor 40 (GPR40)-dependent way. In this study, we questioned whether higher doses of GW9508 may also influence resorbing cell viability specifically. Interestingly, GW9508 at 100 µM altered osteoclast precursor (OcP) viability while it had positive effects on osteoblastic precursors suggesting an activity dependent on the cell lineage. According to 7-AAD/Annexin-V staining, induced OcP cell death was found to be associated with necrosis mechanisms. Consistently, GW9508 led to a sustained establishment of oxidative stress from mitochondrial origin. In contrast to previous observations on osteoclast differentiation inhibition, OcP viability targeted by high doses of GW9508 appeared to be independent of GPR40 involvement. Although mediating structures remain to be determined, our data demonstrate for the first time that this fatty acid receptor agonist driving OcP specific cell death may now open new perspectives regarding therapeutic strategies in osteolytic disorders.

Does the surgeon's experience influence the outcome of laparoscopic treatment of common bile duct stones?

BACKGROUND: The aim of this study was to assess laparoscopic treatment of choledocholithiasis with respect to the surgeon's experience. METHODS: From January 1994 to December 2006, 130 patients underwent laparoscopic treatment for common bile duct stones found with intraoperative cholangiography. Two types of surgeons were defined: junior surgeons with fewer than ten laparoscopic common bile duct explorations performed and experienced surgeons with more than ten. The two patient populations (n = 65 in each group) were similar in regard to demographic data, clinical presentations (complicated or not), and ASA score. RESULTS: Results show that junior surgeons had significantly more patients with a common bile duct (CBD) diameter <7 mm compared to experienced surgeons (66% vs. 38%; p = 0.002). Primary closure of choledochotomy was performed by senior rather than junior surgeons significantly more often (87.5% vs. 69%; p = 0.05). Mean operating time was found to be longer for junior operators than for experienced surgeons (220 ± 71 min vs. 169 ± 71 min; p = 0.0006). There was no difference between group 1 (juniors) and group 2 (experienced surgeons) in regard to laparotomy conversion rate (9% vs. 1.5%; p = 0.1), complete common bile duct clearance (98% vs. 100%, p = ns), postoperative complications (two bile leaks in group 1 and one in group 2), and hospital stay (9 days vs. 7.5 days). In multivariate analysis, the transcystic approach was not influenced by the surgeon's experience. Experienced surgeons performed choledochotomy with primary closure more easily [RR = 3 (range = 1.1-8); p = 0.04]. Complicated presentations [RR = 2 (0.7-3); p = 0.08] and CBD diameter [RR = 2.5 (0.96-7); p = 0.06] influenced the choice of type of closure of choledochotomy without any significant value. CONCLUSION: Surgeon's experience influenced operating time and type of choledochotomy closure performed but had no influence on postoperative results of the laparoscopic treatment of common bile duct stones.

The Internal Conduit System of the Swine Inverted Lymph Node.

Lymph nodes (LN) are the crossroad where naïve lymphocytes, peripheral antigens and antigen presenting cells contact together in order to mount an adaptive immune response. For this purpose, LN are highly organized convergent hubs of blood and lymphatic vessels that, in the case of B lymphocytes, lead to the B cell follicles. Herein take place the selection and maturation of B cell clones producing high affinity antibodies directed against various antigens. Whereas the knowledge on the murine and human LN distribution systems have reached an exquisite precision those last years, the organization of the antigens and cells circulation into the inverted porcine LN remains poorly described. Using up to date microscopy tools, we described the complex interconnections between afferent lymphatics and blood vessels, perifollicular macrophages, follicular B cells and efferent blood vessels. We observed that afferent lymphatic sinuses presented an asymmetric Lyve-1 expression similar to the one observed in murine LN, whereas specialized perifollicular sinuses connect the main afferent lymphatic sinus to the B cell follicles. Finally, whereas it was long though that mature B cells egress from the inverted LN in the T cell zone through HEV, our observations are in agreement with mature B cells accessing the efferent blood circulation in the efferent, subcapsular area. This understanding of the inverted porcine LN circuitry will allow a more accurate exploration of swine pathogens interactions with the immune cells inside the LN structures. Moreover, the mix between similarities and differences of porcine inverted LN circuitry with mouse and human normal LN shall enable to better apprehend the functions and malfunctions of normal LN from a new perspective.

GPR40 mediates potential positive effects of a saturated fatty acid enriched diet on bone.

SCOPE: The stimulation of the free fatty acid receptor G-protein coupled receptor (GPR) 40 by GW9508 prevents bone loss by inhibiting osteoclast activity, both in vitro and in vivo. Here, we questioned whether the stimulation of the GPR40 receptor by dietary fatty acids may lead to the same beneficial effect on bone. METHODS AND RESULTS: We investigated (i) the impact of a fatty acid enriched diet (high-fat diet [HFD]) on bone health in C57/BL6 female mice depending on (ii) the estrogen status (ovariectomy) and (iii) the genotype (GPR40+/+ or GPR40-/- ). Bone mineral density (BMD), body composition, weight, inflammation and bone remodeling parameters were monitored. HFD decreased BMD in HFD-SH-GPR40+/+ mice but OVX failed to further impact BMD in HFD-OVX-GPR40+/+ mice, while additional bone loss was observed in HFD-OVX-GPR40-/- animals. These data suggest that when stimulated by fatty acid enriched diets GPR40 contributes to counteract ovariectomy-induced bone alteration. The sparing effect is supported by the modulation of both the osteoprotegerin/receptor activator of nuclear factor kappa-B ligand (OPG/RANKL) ratio in blood stream and the expression level of inflammatory markers in adipose tissues. Bone preservation by GPR40 stimulation is dependent on the presence of long-chain saturated fatty acids. CONCLUSION: GPR40 contributes to counter ovariectomy-induced bone loss in a context of saturated fatty acid enrichment.

Deficiency of G-protein coupled receptor 40, a lipid-activated receptor, heightens in vitro- and in vivo-induced murine osteoarthritis.

Osteoarthritis (OA) is an age-related degenerative joint disease. To date, its management is focused on symptoms (pain and inflammation). Studies suggest that fatty acids can reduce the expression of inflammatory and catalytic mediators, and improve in vivo joint function. Free fatty acid receptors (FFARs) such as G-protein coupled receptor 40 (GPR40) are proposed as attractive therapeutic targets to counteract inflammation and cartilage degradation observed in OA. This study aims to elucidate the involvement of GPR40 in OA. In this study, we used an in vitro model of OA, and surgically induced OA by ligament transection and partial meniscectomy in wild-type and GPR40 deficient mice. OA phenotype was investigated in vivo by histology and genes expression. We demonstrate that IL-1ß-treated GPR40(-/-) chondrocytes secret more inflammatory mediators (nitric oxide, interleukin-6, prostaglandin E2) and active catabolic enzymes (metalloproteinase-2, -9 [MMP-2, MMP-9]), and show decreased anabolism (glycoaminoglycan) compared to GPR40(+/+) cells. In accordance with these results, we show that GPR40(-/-) mice exhibit an aggravated OA-induced phenotype characterized by higher tidemark exposure, frequency of osteophyte formation and subchondral bone sclerosis. Altogether our results demonstrate that GPR40 deficiency leads to an extended OA phenotype, providing evidence that increasing GPR40 activity, by natural or synthetic ligands, could be a new strategy in the management of OA.

Pros and cons of fatty acids in bone biology.

Despite the growing interest in deciphering the causes and consequences of obesity-related disorders, the mechanisms linking fat intake to bone behaviour remain unclear. Since bone fractures are widely associated with increased morbidity and mortality, most notably in elderly and obese people, bone health has become a major social and economic issue. Consistently, public health system guidelines have encouraged low-fat diets in order to reduce associated complications. However, from a bone point of view, mechanisms linking fat intake to bone alteration remain quite controversial. Thus, after more than a decade of dedicated studies, this timely review offers a comprehensive overview of the relationships between bone and fatty acids. Using clinical evidences as a starting-point to more complex molecular elucidation, this work highlights the complexity of the system and reveals that bone alteration that cannot be solved simply by taking ω-3 pills. Fatty acid effects on bone metabolism can be both direct and indirect and require integrated investigations. Furthermore, even at the level of a single cell, one fatty acid is able to trigger several different independent pathways (receptors, metabolites…) which may all have a say in the final cellular metabolic response.

Have French recommendations had an impact on the treatment and survival of middle and lower rectal cancer patients?

OBJECTIVE: Compare the survival of middle and lower rectal cancer (MLRC) patients before and after the 1994 issue of rectal cancer (RC) consensus conference recommendations. METHODS: Cases of MLRC noted in the Hérault department of France in 1992 (n=58) and 2000 (n=93) yielded exhaustive epidemiological, clinical-pathological and treatment data that were used to compare MLRC patient management and survival in these two periods. RESULTS: Significantly more lymph nodes (≥ 8) were harvested in 2000 (≥ 8, 47%) than in 1992. In all, 45 patients (77.6%) received radiotherapy in 1992, and 74 (82%) in 2000. Chemotherapy was employed in 15 patients (25.9%) in 1992 and in 39 patients (43%) in 2000. Chemotherapy and radiotherapy, together with sphincter conservation, were dependent upon the year. Overall 5-year relative survival for rectal cancer in the Hérault department did not vary between 1992 (56%) and 2000 (56%). Independent poor prognostic factors were the same in both years: age over 75 years, lymph node involvement and metastases. Management place and year had no significant impact on prognosis. CONCLUSION: The recommendations made have had little impact on disease management and the quality of anatomic pathology reports, and have not improved 5-year relative survival.

Borage and fish oils lifelong supplementation decreases inflammation and improves bone health in a murine model of senile osteoporosis.

Fats are prevalent in western diets; they have known deleterious effects on muscle insulin resistance and may contribute to bone loss. However, relationships between fatty acids and locomotor system dysfunctions in elderly population remain controversial. The aim of this study was to analyze the impact of fatty acid quality on the age related evolution of the locomotor system and to understand which aging mechanisms are involved. In order to analyze age related complications, the SAMP8 mouse strain was chosen as a progeria model as compared to the SAMR1 control strain. Then, two months old mice were divided in different groups and subjected to the following diets : (1) standard "growth" diet - (2) "sunflower" diet (high ω6/ω3 ratio) - (3) "borage" diet (high γ-linolenic acid) - (4) "fish" diet (high in long chain ω3). Mice were fed ad libitum through the whole protocol. At 12 months old, the mice were sacrificed and tissues were harvested for bone studies, fat and muscle mass measures, inflammation parameters and bone cell marker expression. We demonstrated for the first time that borage and fish diets restored inflammation and bone parameters using an original model of senile osteoporosis that mimics clinical features of aging in humans. Therefore, our study strongly encourages nutritional approaches as relevant and promising strategies for preventing aged-related locomotor dysfunctions.