Multi-Inlet Spheroid Generator for High-Throughput Combinatorial Drug Screening Based on the Tumor Microenvironment.

Tumor spheroids are powerful tools for drug screening and understanding tumor physiology. Among spheroid formation methods, the hanging drop method is considered most suitable for high-throughput screening (HTS) of anticancer drugs because it does not require surface treatment. However, it still needs to increase the liquid-holding capacity because hanging drops often fall due to the increased pressure caused by the addition of drugs, cells, etc. Here, we report a multi-inlet spheroid generator (MSG) enabling the stable addition of liquid-containing drugs or cells into a spheroid through its side inlet. The MSG was able to load additional solutions through the side inlet without increasing the force applied to the hanging drop. The volume of the additional liquid was easily controlled by varying the diameter of the side inlet. Furthermore, the sequences of the solution injections were manipulated using multiple side inlets. The feasibility of the MSG in clinical application was demonstrated by testing the efficacy of drugs in patient-derived cancer (PDC) cells and controlling the stromal cell ratio in the tumor microenvironment (TME) containing spheroids. Our results suggest that the MSG is a versatile platform for HTS of anticancer drugs and recapitulating the TME.

Efficacy of a local anesthetic gel infusion kit for pain relief after minimally invasive colorectal surgery: an open-label, randomized clinical trial.

Continuous wound infusion with local anesthesia is an effective method for reducing postoperative pain after laparoscopic colorectal surgery. However, most subcutaneous local anesthesia is delivered through continuous injection, which can be inconvenient for patients. This study compared the effectiveness of postoperative pain relief from the application of a local poloxamer 407-based ropivacaine hydrogel (Gel) to the incision site with continuous infusion-type ropivacaine administration (On-Q) in patients undergoing laparoscopic colorectal surgery. This prospective, randomized, non-inferiority study included 61 patients who underwent laparoscopic colorectal surgery with an incision length of 3-6 cm. All 61 patients were randomly assigned to the Gel group (poloxamer 407-based 0.75% ropivacaine, 22.5 mg) or the On-Q group (0.2% ropivacaine, 4 mg/hour for two days). Postoperative analgesia was induced in all patients with intravenous patient-controlled analgesia (IV-PCA). The outcome measures, which were assessed for 72 h after surgery, included the total amount of fentanyl consumed via IV-PCA (primary endpoint), and the amount of rescue analgesia (pethidine) and postoperative pain intensity assessed using a numeric rating scale (NRS) [secondary endpoints]. The Gel was administered to 31 patients and On-Q was used for 30 patients. There was no significant difference in the total usage of fentanyl between the two groups (Gel group, 1623.98 mcg; On-Q group, 1595.12 mcg; P = 0.806). There was also no significant difference in the frequency of analgesic rescue medication use (P = 0.213) or NRS scores (postoperative 6 h, P = 0.860; 24 h, P = 0.333; 48 h, P = 0.168; and 72 h, P = 0.655) between the two groups. The Gel, which continuously delivers a local anesthetic to operative sites, can thus be considered an effective device for analgesia and pain relief for midline incisions in laparoscopic colorectal surgery.

Bioactive molecular network-guided discovery of dihydro-ß-agarofurans from the fruits of Celastrus orbiculatus.

A bioactive molecular networking strategy has been applied to discovery of bioactive constituents from the fruits of Celastrus orbiculatus Thunb., which showed significant inhibitory effects on the α-MSH-induced melanin production in B16F0 melanoma cells. In the obtained molecular network, the nodes with relatively high bioactive scores were prioritized for isolation; as a result, 12 undescribed dihydro-ß-agarofuran sesquiterpenes together with 15 known compounds were isolated from MeOH extracts of the fruits of C. orbiculatus. Their structures were elucidated based on the interpretation of NMR, HRESIMS, ECD data, and single crystal X-ray diffraction. Among the obtained isolates, celastorbin A and (1R,2S,4R,5S,7S,8S,9R,10S)-1,2,8-triacetoxy-9-cinnamoyloxydihydro-ß-agarofuran, which possessed high bioactive scores in the molecular network, exhibited potent inhibitory effects on the α-MSH-induced melanin production in B16F0 cells with IC50 values of 4.1 and 2.0 µM, respectively.

Chemical constituents from Pterocarpus santalinus and their inhibitory effects on nitric oxide production.

A tropolone (2) and an acorane sesquiterpene (3), along with twenty previously known compounds were isolated from the heartwood of Pterocarpus santalinus. The structure of the isolated compounds was elucidated via 1D and 2D NMR spectroscopy and HRESIMS analysis. The absolute configuration of 3 was determined by comparison of the experimental and calculated ECD data. All compounds were evaluated for their inhibitory effects against nitric oxide production in LPS-stimulated RAW 264.7 macrophages.

Sesquiterpenoids from Chrysanthemum indicum with Inhibitory Effects on NO Production.

Three new guaianolide lactones (1-3) and four new 9-oxonerolidol glucosides (5-8) together with 20 known compounds were isolated from the MeOH extract of the flowers of Chrysanthemum indicum. Their structures were elucidated based on the interpretation of NMR, HRESIMS, and electronic circular dichroism (ECD) data along with acid hydrolysis. Of the isolates, sesquiterpenoids 1-4 and 15 and flavones 17 and 18 exhibited inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide production in RAW 264.7 cells with IC50 values in the range 0.2-27.0 µM.

Clinical and molecular distinctions in patients with refractory colon cancer who benefit from regorafenib treatment.

Regorafenib (Stivarga, BAY 73-4506; Bayer Pharma AG, Berlin, Germany) is a novel oral multikinase inhibitor that blocks the activity of several protein kinases. However, few guidelines exist for novel biomarkers to select patients who will likely benefit from regorafenib treatment. Metastatic colorectal cancer (mCRC) patients treated with regorafenib were evaluated in this study. Tumor tissues of these patients were subjected to next-generation sequencing-based cancer panel tests. The relationship between molecular profiling and efficacy of regorafenib was analyzed. Among the 76 mCRC patients, the median age was 58 years (range 22-79 years), and 73.7% received regorafenib as a third-line therapy. The primary tumor locations were the right side (n = 15, 19.8%) and the left side (n = 61, 80.2%). Most patients (97.4%) had received prior anti-angiogenetic agents, and a prior anti-Epidermal Growth Factor Receptor (EGFR) agent had been administered to 32.9%. Of these 76 patients, 65 were evaluated to determine the efficacy of treatment. We observed zero complete responses, seven confirmed partial responses (PR 9.2%), 26 stable disease states (34.2%), and 32 disease progressions (42.1%). The overall confirmed response rate and the disease control rate were 9.2% and 43.4%, respectively. Genomic analysis revealed that APC mutations were significant in patients who demonstrated a tumor response to regorafenib (p < 0.05). Interestingly, FGFR1 amplification was detected in only three of 76 patients (3.9%), and these three patients achieved a PR to regorafenib. The median progression-free survival time was 2.8 months (95% Confidence Interval [CI] 1.6-4.0). Patients with BRAF mutation and/or SMAD4 mutation had significantly worse progression-free survival (PFS) than those without such a mutation. On pathway analysis, Tumor Growth Factor (TGF)-beta pathways were significantly associated with worse PFS. We found that efficacy of regorafenib might be correlated with specific genetic aberrations, such as APC mutation and FGFR1 amplification. In addition, SMAD4 mutation and TGF-beta pathway were associated with worse PFS after regorafenib. We found that efficacy of regorafenib might be correlated with specific genetic aberrations, such as APC mutation and FGFR1 amplification. In addition, SMAD4 mutation and the TGF-beta pathway were associated with worse PFS after regorafenib.

Protective Effect of Ginkgo Biloba Extract on Gentamicin-Induced Structural Changes of Calcite-Gelatin Composite.

OBJECTIVE: To evaluate the protective effect of Ginkgo biloba extract (GBE) on gentamicin (GM)-induced morphological damage of an artificial otoconia. MATERIALS AND METHODS: An artificial otoconia powder was placed in a 12-well culture plate containing artificial endolymph. GM (500 µL; 40 mg/mL) was added to the first four wells. GM (500 µL; 40 mg/mL) with GBE (500 µL) was added to the next four wells. PBS (500 µL) was added to the remaining four wells as a control. The levels of nitric oxide (NO) synthesis were determined in the supernatants of each group. Alteration in surface morphology and calcium content were determined by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX), respectively. RESULTS: NO concentration increased in the GM-treated group compared with that in the control group. When the calcite-gelatin composite was treated with GBE, GM-stimulated NO production significantly decreased. The surface of the calcite-gelatin composite exposed to GM showed erosive fissures and had a honeycomb-like appearance. GBE showed a protective effect against dissolution. EDX showed that the calcium content of the GM-treated group significantly decreased. However, the GBE-treated group demonstrated an insignificant change in the calcium concentration compared with the control. CONCLUSION: From these results, we can conclude that GBE may have a protective effect against GM-induced NO production and superficial structural changes.

ALH-L1005 attenuates endotoxin induced inner ear damage.

OBJECTIVE: To assess whether this compound (ALH-L1005) is conceivably an effective agent in protecting against cochlear damage induced by LPS. MATERIALS AND METHODS: Tube formation using human umbilical vein endothelial cell (HUVEC) and matrix metalloproteinase (MMP)-9 inhibition assay was performed. 24 guinea pigs were randomly divided into three groups. Intratympanic instillation of LPS (n=8) as negative control, instillation of oxytetracycline 1h after LPS as positive control (n=8), and intratympanic instillation of ALH-L1005 (n=8) 1h after LPS were considered experimental group. Evaluation by auditory brainstem response (ABR) measurement, cochlear blood flow, and blood-labyrinth barrier (BLB) permeability were performed. Cochlear hair cells were observed by field emission-scanning electron microscopy (FE-SEM). MMP-9 activation was measured by gelatin zymography. RESULTS: For HUVEC, the tube formation was suppressed in a dose dependant manner. ALH-L1005 inhibited the MMP-9 activity prominently. It also attenuated the elevation of LPS-induced hearing threshold shift and recovery of CBF. By FE-SEM, cochlear hair cells could be preserved in experimental group. ALH-L1005 significantly reduced the BLB opening compared to LPS group. Active MMP-9 expression could be detected in the LPS group. In contrast to ALH-L1005 group, active MMP-9 expression was not detected. CONCLUSION: Our results conclude that ALH-L1005 showed a protective effect in the cochlear lateral wall damage induced by LPS.

Prognostic factors associated with primary cancer in curatively resected stage IV colorectal cancer.

PURPOSE: The aim of this study is to evaluate the prognostic factors associated with primary cancer in patients with curatively resected stage IV colorectal cancer, based on lymph node status. METHODS: A total of 468 consecutive patients with curatively resected stage IV colorectal cancer from October 1994 to December 2010 were prospectively enrolled. Survival curves were constructed using the Kaplan-Meier method, and multivariate analysis assessed independent prognostic factors. RESULTS: During the median follow-up period of this study, which was 37 months (range 1-177), the 3- and 5-year overall survival rates were 66.5 and 52.1%, respectively, and the 3- and 5-year disease-free survival rates were 43.0 and 38.2%, respectively. According to multivariate analysis, adjuvant chemotherapy and the preoperative serum carcinoembryonic antigen (CEA) level were independent prognostic factors for overall survival, and primary tumor location and preoperative serum CEA level were independent variables for disease-free survival. For the patients with N0 and N1 tumors, the overall survival curves in the preoperative CEA groups differed significantly (P = 0.046 and P < 0.013, respectively). However, for patients with pN2 tumors, the overall survival did not differ significantly according to the preoperative CEA (P = 0.948). CONCLUSION: The preoperative serum CEA level is a reliable predictor of recurrence and survival after curative surgery in patients with metastatic colorectal cancer. A multidisciplinary approach that combines both complete resection and adjuvant chemotherapy may achieve improved overall survival in these patients.

Growth rate, organic carbon and nutrient removal rates of Chlorella sorokiniana in autotrophic, heterotrophic and mixotrophic conditions.

This study sought to investigate the growth rate and organic carbon and nutrient removal efficiency of Chlorella sorokiniana under autotrophic, heterotrophic and mixotrophic conditions. Growth rates of the microalgae were 0.24 d(-1), 0.53 d(-1) and 0.44 d(-1) in autotrophic, heterotrophic and mixotrophic conditions, respectively. The growth rate of C. sorokiniana was significantly higher for that grown under heterotrophic conditions. The nitrogen removal rates were 13.1 mg-N/L/day, 23.9 mg-N/L/day and 19.4 mg-N/L/day, respectively. The phosphorus removal rates reached to 3.4 mg-P/L/day, 5.6 mg-P/L/day and 5.1 mg-P/L/day, respectively. Heterotrophic conditions were superior in terms of the microalgae growth and removal of nitrogen and phosphorus compared to autotrophic and mixotrophic conditions, suggesting that microalgae cultured under this condition would be most useful for application in wastewater treatment systems.

Mastoid obliteration using three-dimensional composite scaffolds consisting of polycaprolactone/ß-tricalcium phosphate/collagen nanofibers: an in vitro and in vivo study.

Two different composite scaffolds, solid-freeform-fabricated PCL/ß-TCP supplemented with and without collagen nanofibers are fabricated. These scaffolds are evaluated whether a combination of collagen nanofibers with PCL/ß-TCP can promote osteogenesis in a mastoid obliteration. To assess the effects of the cellular activities of osteoblast-like-cells (MG63), SEM images and MTT assays are conducted. Experimental mastoid obliteration is performed using guinea pigs that are divided group A (PCL/ß-TCP/collagen-nanofiber scaffold) and group B (PCL/ß-TCP scaffold). The results reveal that PCL/ß-TCP/collagen scaffold provide much broader cell attachment sites than PCL/ß-TCP scaffold. The µ-CT and fluorescent microscopy results reveal that the acceleration of early new bone formation within the pores and scaffold itself at week 4 post-operation is more effective in group A. In addition, based on the results of the histological and µ-CT at 12 weeks post-surgery, the effective regeneration of bone in the PCL/ß-TCP/collagen scaffold is appeared.

Effect of ginkgo biloba extract on recovery after facial nerve crush injury in the rat.

BACKGROUND AND OBJECTIVE: Many pharmacological agents have shown successful results in experimental crush injury of the peripheral nerve. To date, therapeutic effect of ginkgo biloba extract (GBE) on the peripheral nerve crush injury of rats has been rarely reported, moreover, neuroprotective effect on the facial nerve crush injury has not been reported. MATERIALS AND METHODS: Prospective functional recovery, using a vibrissae movement and electrophysiological analysis of recovery 4 weeks after the facial nerve crush in adult rats, and comparison with randomized intraperitoneal injection of either GBE or control phosphate buffered saline. RESULTS: Relative to the control group (26 days post operation), administration of GBE significantly accelerated the recovery of vibrissae orientation to 11.7 days post the operation. A significant functional recovery was observed by postoperative 2nd week in the experimental group. The recovery of threshold and conduction velocity, postoperative 4th week in the experimental group, showed statistically significant difference compared to that of the control group. CONCLUSION: From this result, intraperitoneal injection of GBE has been found effective in promoting the regeneration of the nerve in an experimental facial nerve crush rat model. Further studies, including morphological and molecular analyses, are necessary to clarify the mechanisms of GBE on the facial nerve crush.

The effect of postoperative pelvic irradiation after complete resection of metastatic rectal cancer.

BACKGROUND AND OBJECTIVES: The 2010 NCCN clinical practice guidelines recommend radiation as a part of the standard adjuvant or neoadjuvant treatment for stage IV rectal cancer patients. This study evaluated the oncologic efficacy of postoperative radiotherapy (RTx) in loco-regional control after complete removal of primary and metastatic lesions in stage IV rectal cancer. METHODS: Sixty-eight patients with metastatic rectal cancer were enrolled and analyzed. Twenty-eight of the enrolled patients received concurrent postoperative RTx with chemotherapy (RTx group) and the remaining 40 received only postoperative systemic chemotherapy (CTx) without RTx (non-RTx group). The eligibility criteria were as follows: a primary rectal tumor located in the low or mid-rectum, no postoperative macroscopic and microscopic evidence of residual tumor in primary and metastatic sites, and no history of prior CTx or pelvic RTx. RESULTS: The recurrence rates were 75.0% in the RTx group and 72.5% in the non-RTx group. Local recurrence rates were 7.1% (RTx group) and 22.5% (non-RTx group) (P = 0.108). There were no differences in overall survival (OS), local recurrence-free survival, and disease-free survival between the two groups. The 2-year OS rates were 78.9% (RTx group) and 74.1% (non-RTx group) (P = 0.395). CONCLUSIONS: Survival benefit of postoperative RTx in stage IV rectal cancer after complete removal of tumors was not apparent. RTx could be recommended for selected patients at high risk of local recurrence or for palliation of symptoms.

Clinical impact of microsatellite instability in colon cancer following adjuvant FOLFOX therapy.

PURPOSE: Colon cancer with DNA mismatch repair (MMR) defects reveals indistinguishable clinical and pathologic aspects, including better prognosis and reduced response to 5-fluorouracil (5-FU)-based chemotherapy. There has been no consensus for p53 as a prognostic marker in colorectal cancer. This study investigated the clinical implication of MSI-H/MMR-D and p53 expression in R0-resected colon cancer patients who received adjuvant oxaliplatin/5-FU/leucovorin (FOLFOX) therapy. EXPERIMENTAL DESIGN: We analyzed 135 patients, who had been treated by adjuvant chemotherapy containing 5-FU and oxaliplatin (FOLFOX) after curative resection (R0) for colon adenocarcinoma between May 2004 and November 2007. Tumor expression of the MMR proteins, MLH1 and MSH2, was detected by immunohistochemistry (IHC) in surgically resected tumor specimens. MSI was analyzed by polymerase chain reaction (PCR) amplification using fluorescent dye-labeled primers specific for microsatellite loci. Tumors with MMR defects were defined as those demonstrating loss of MMR protein expression (MMR-D) and/or microsatellite instability high (MSI-H) genotype. Expression patterns of p53 were determined in a semiquantitative manner by light microscopy. RESULTS: There were 13 (9.6%) patients with stage II, 108 (80%) with stage III, and 14 (10.4%) with stage IV. Fourteen patients with stage IV (10.3%) had metastases to liver only, all of whom underwent complete metastasectomy for liver metastases. In total, 134 tumor specimens were genotyped, 115 specimens were tested by IHC and 113 cases had both genotyping and IHC results available for analysis. Genotyping results demonstrated that 12 (9.0%) cases were MSI-H and 122 (91.0%) were MSI-L/S. By IHC, 11 (9.6%) patients were MMR-D and 104 (90.4%) were MMR-I. The methods were in agreement in 108 patients (94.7%). We assessed 114 patients for p53 expression by immunostaining. MMR status was not significantly associated with DFS (P = 0.56) or OS (P = 0.61) in patients with colon cancer (n = 135) receiving adjuvant FOLFOX. According to p53 status, there was also no significant difference for DFS (P = 0.11) and OS (P = 0.94). For patients with genotyping/IHC agreement (n = 108), there was no difference in DFS (P = 0.57) and OS (P = 0.98) between patients with MSI-H/MMR-D and MSI-L/S/MMR-I tumors. CONCLUSION: The MMR status or p53 positivity was not significantly associated with outcomes to FOLFOX as adjuvant chemotherapy in colon cancer patients with R0 resection. Adding oxaliplatin in adjuvant chemotherapy may overcome negative impact of 5-FU on colon cancers with MSI-H/MMR-D.

Relationship of polymorphism of the tandem repeat sequence in the thymidylate synthase gene and the survival of stage III colorectal cancer patients receiving adjuvant 5-flurouracil-based chemotherapy.

BACKGROUND: The aim of this study was to determine whether the different polymorphisms in the thymidylate synthase (TS) gene, novel G>C single nucleotide polymorphism (SNP) and variable number of tandem repeat (VNTR), may be related with disease-free survival (DFS) in patients with stage III colorectal cancer receiving adjuvant chemotherapy. METHODS: The study included 201 patients with pathologic TNM stage III colon cancer who received adjuvant 5-fluorouracil (5-FU)-based chemotherapy after surgery. DNA was extracted from fresh tumor tissue and sequenced. Patients with TS genotypes of 2R3G, 3C3G, or 3G3G were assigned to a high expression group, and those with 2R2R, 2R3C, or 3C3C, to a low expression group. RESULTS: Frequencies of the TS tandem repeat polymorphisms among the tumor genotypes were 6.0% in 2R2R, 25.4% in 2R3R, and 68.7% in 3R3R. The low expression group included 52 patients (25.9%), and the high expression group included 149 patients (74.1%). Groups classified according to possession of VNTR, SNP, and low- or high-expression genotypes did not differ significantly in DFS. In multivariate analysis, only tumor stage showed significant prognostic value (hazard ratio (HR) 2.05, 95% CI = 1.24-3.37, P = 0.005). CONCLUSIONS: TS polymorphisms do not predict clinical outcome of colorectal cancer patients treated with adjuvant 5-FU-based chemotherapy.

Tumor localization for laparoscopic colorectal surgery.

BACKGROUND: Because palpating colonic tumors during laparoscopy is impossible, the precise location of a tumor must be identified before operation. The aim of this study was to evaluate the accuracy of various diagnostic methods that are used to localize colorectal tumors and to propose an adequate localization protocol for laparoscopic colorectal surgery. METHODS: A total of 310 patients underwent laparoscopy-assisted colectomy between April 2000 and March 2006. We investigated if the locations of the tumors that were estimated preoperatively were consistent with the actual locations according to the operation. RESULTS: All the tumors were correctly localized and resected. Altogether, 203 patients had complete endoscopic reports available. Colonoscopy was inaccurate for tumor localization in 23 cases (11.3%). In total, 104 patients (33.5%) underwent barium enema; five tumors (4.8%) were not visualized, and three tumors were incorrectly localized. Another group of 94 patients (30.3%) underwent computed tomography (CT) colonography, which identified 91 of 94 lesions (96.8%). Finally, 96 patients (31.0%) underwent endoscopic tattooing; 2 patients (2.1%) did not have tattoos visualized laparoscopically and required intraoperative colonoscopy to localize their lesions during resection. Dye spillage was found in six patients intraoperatively, but only one patient experienced clinical symptoms. Intraoperative colonoscopy was performed in four patients; two of the four were followed by endoscopic tattooing, and the other two underwent intraoperative colonoscopy for localization. All lesions were correctly localized by intraoperative colonoscopy. The accuracy of tumor localization was as follows: colonoscopy (180/203, 88.7%), barium enema (97/104, 93.3%), CT colonography (89/94, 94.7%), endoscopic tattooing (94/96, 97.9%), and intraoperative colonoscopy (4/4, 100%). CONCLUSIONS: With a combination of methods, localization of tumors for laparoscopic surgery did not seem very different from that during open surgery. Preoperative endoscopic tattooing is a safe, highly effective method for localization. In the case of tattoo failure, intraoperative colonoscopy can be used for accurate localization.