AIMTo establish a rat model of anal sphincter injury and test different systems to provide stem cells to injured area.METHODSAdipose-derived stem cells (ASCs) were isolated from BDIX rats and were transfected with green fluorescent protein (GFP) for cell tracking. Biosutures (sutures covered with ASCs) were prepared with 1.5 x 106 GFP-ASCs, and solutions of 106 GFP-ASCs in normal saline were prepared for injection. Anorectal normal anatomy was studied on Wistar and BDIX female rats. Then, we designed an anal sphincter injury model consisting of a 1-cm extra-mucosal miotomy beginning at the anal verge in the anterior middle line. The sphincter lesion was confirmed with conventional histology (hematoxylin and eosin) and immunofluorescence with 4', 6-diamidino-2-phenylindole (commonly known as DAPI), GFP and α-actin. Functional effect was assessed with basal anal manometry, prior to and after injury. After sphincter damage, 36 BDIX rats were randomized to three groups for: (1) Cell injection without repair; (2) biosuture repair; and (3) conventional suture repair and cell injection. Functional and safety studies were conducted on all the animals. Rats were sacrificed after 1, 4 or 7 d. Then, histological and immunofluorescence studies were performed on the surgical area.RESULTSWith the described protocol, biosutures had been covered with at least 820000-860000 ASCs, with 100% viability. Our studies demonstrated that some ASCs remained adhered after suture passage through the muscle. Morphological assessment showed that the rat anal anatomy is comparable with human anatomy; two sphincters are present, but the external sphincter is poorly developed. Anal sphincter pressure data showed spontaneous, consistent, rhythmic anal contractions, taking the form of “plateaus” with multiple twitches (peaks) in each pressure wave. These basal contractions were very heterogeneous; their frequency was 0.91-4.17 per min (mean 1.6980, SD 0.57698), their mean duration was 26.67 s and mean number of peaks was 12.53. Our morphological assessment revealed that with the aforementioned surgical procedure, both sphincters were completely sectioned. In manometry, the described activity disappeared and was replaced by a gentle oscillation of basal line, without a recognizable pattern. Surprisingly, these findings appeared irrespective of injury repair or not. ASCs survived in this potentially septic area for 7 d, at least. We were able to identify them in 84% of animals, mainly in the muscular section area or in the tissue between the muscular endings. ASCs formed a kind of “conglomerate” in rats treated with injections, while in the biosuture group, they wrapped the suture. ASCs were also able to migrate to the damaged zone. No relevant adverse events or mortality could be related to the stem cells in our study. We also did not find unexpected tissue growths.CONCLUSIONThe proposed procedure produces a consistent sphincter lesion. Biosutures and injections are suitable for cell delivery. ASCs survive and are completely safe in this clinical s...

Biosutures are a comfortable way of stem cell delivery to the surgical field without modification of the operative protocol. ASCs suppress the local acute inflammatory reaction (increased macrophage migration and decreased neutrophil infiltration) in the tracheal anastomosis and cause an early switch from acute to chronic inflammation.

The usefulness of local collagenase in therapeutic approaches to solid tumors has been tested recently. In this study, we evaluate the safety and efficacy of intraperitoneal collagenase associated or not to mitomycin for treatment of colorectal peritoneal metastases in an experimental rat model. Using a fixed-dose procedure, we found that a dose of collagenase of 37 IU/mL administered for 15 min with a hyperthermia pump at 37.5 °C, both in isolation or associated to sequential treatment with intraperitoneal mitomycin, led to a macroscopic decrease in tumor volume as evaluated by the modified peritoneal cancer index (mPCI). Concerning the safety of the procedure, the animals showed no physiological or behavioral disorders during 8 weeks of follow-up. Local treatment for peritoneal metastases of colorectal origin with intraperitoneal collagenase has proved safe and effective in an experimental murine model. Therefore, the stroma-first approach by enzymatic breakdown of collagen from the tumor's extracellular matrix provides a new therapeutic target for colorectal peritoneal metastases.

In vitro studies of partially purified virtosomes from rat liver showed inhibition of cell multiplication in four normal and two tumour cell lines. In vivo, the liver virtosomes slowed tumour growth and limited metastases in rats bearing DHD/K12-PROb cell initiated tumours.

AIMTo assess KRAS G12D mutation detection by droplet digital PCR (ddPCR) in stool-derived DNA from colorectal cancer (CRC) patients.METHODSIn this study, tumor tissue and stool samples were collected from 70 patients with stage I-IV CRC diagnosed by preoperative biopsy. KRAS mutational status was determined by pyrosequencing analysis of DNA obtained from formalin-fixed paraffin-embedded (FFPE) tumor tissues. The KRAS G12D mutation was then analyzed by ddPCR in FFPE tumors and stool-derived DNA from patients with this point mutation. Wild-type (WT) tumors, as determined by pyrosequencing, were included as controls; analysis of FFPE tissue and stool-derived DNA by ddPCR was performed for these patients as well.RESULTSAmong the total 70 patients included, KRAS mutations were detected by pyrosequencing in 32 (45.71%), whereas 38 (54.29%) had WT tumors. The frequency of KRAS mutations was higher in left-sided tumors (11 located in the right colon, 15 in the left, and 6 in the rectum). The predominant point mutation was KRAS G12D (14.29%, n = 10), which was more frequent in early-stage tumors (I-IIA, n = 7). In agreement with pyrosequencing results, the KRAS G12D mutation was detected by ddPCR in FFPE tumor-derived DNA, and only a residual number of mutated copies was found in WT controls. The KRAS G12D mutation was also detected in stool-derived DNA in 80% of all fecal samples from CRC patients with this point mutation.CONCLUSIONddPCR is a reliable and sensitive method to analyze KRAS G12D mutation in stool-derived DNA from CRC patients, especially at early stages. This non-invasive approach is potentially applicable to other relevant biomarkers for CRC management.