CLE3 and its homologs share overlapping functions in the modulation of lateral root formation through CLV1 and BAM1 in Arabidopsis thaliana.

Lateral roots are important for a wide range of processes, including uptake of water and nutrients. The CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION-RELATED (CLE) 1 ~ 7 peptide family and their cognate receptor CLV1 have been shown to negatively regulate lateral root formation under low-nitrate conditions. However, little is known about how CLE signaling regulates lateral root formation. A persistent obstacle in CLE peptide research is their functional redundancies, which makes functional analyses difficult. To address this problem, we generate the cle1 ~ 7 septuple mutant (cle1 ~ 7-cr1, cr stands for mutant allele generated with CRISPR/Cas9). cle1 ~ 7-cr1 exhibits longer lateral roots under normal conditions. Specifically, in cle1 ~ 7-cr1, the lateral root density is increased, and lateral root primordia initiation is found to be accelerated. Further analysis shows that cle3 single mutant exhibits slightly longer lateral roots. On the other hand, plants that overexpress CLE2 and CLE3 exhibit decreased lateral root lengths. To explore cognate receptor(s) of CLE2 and CLE3, we analyze lateral root lengths in clv1 barely any meristem 1(bam1) double mutant. Mutating both the CLV1 and BAM1 causes longer lateral roots, but not in each single mutant. In addition, genetic analysis reveals that CLV1 and BAM1 are epistatic to CLE2 and CLE3. Furthermore, gene expression analysis shows that the LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) genes, which promote lateral root formation, are upregulated in cle1 ~ 7-cr1 and clv1 bam1. We therefore propose that CLE2 and CLE3 peptides are perceived by CLV1 and BAM1 to mediate lateral root formation through LBDs regulation.

Calreticulin exposure induced by anticancer drugs is associated with the p53 signaling pathway in colorectal cancer cells.

Immunogenic cell death (ICD) enhances immunogenicity and activates antitumor immune responses. ICD induction by anticancer drugs may be effective against microsatellite-stable colorectal cancers (CRCs) that are less responsive to immune checkpoint inhibitors. Calreticulin (CRT) is crucial in ICD, promoting dendritic cell phagocytosis and initiating antitumor immunity. This study investigated CRT exposure mechanisms in four CRC cell lines and three human CRC organoids. Flow cytometry and immunofluorescence showed that oxaliplatin and 5-fluorouracil caused CRT exposure in all models. Despite CRT's association with endoplasmic reticulum stress, Western blot analysis showed no increase in this stress. These findings suggest alternative pathways. RNA sequencing identified enrichment of p53 signaling pathway genes, including TP53I3, TP53INP1, and YPEL3, which were confirmed by RT-qPCR. These results suggest that the p53 signaling pathway plays an important role in CRT exposure induced by anticancer drugs.

[A Case of Laparoscopic Abdominoperineal Resection and Perineal Reconstruction for Signet-Ring Cell Carcinoma with Skin Invasion Derived from the Anal Gland].

A 56-year-old man was referred to our hospital with an awareness of anal tumor. The tumor extended from the anal verge to the back of left testicle. Colonoscopy showed no tumor in the rectum and the anal canal. Biopsy showed mucus- producing adenocarcinoma(sig), and we diagnosed anal canal adenocarcinoma with immunostaining. Laparoscopic abdominoperineal rectal resection and perineal reconstruction with the V-Y fasciocutaneous flap closure technique. The patient had no major postoperative complications, and was discharged on 23rd postoperative day. Pathological examination revealed that the tumor was pT3N0M0, pStage ⅡB. The patient received adjuvant chemotherapy with CAPOX and has survived 12 months without recurrence. Immunostaining may be used to diagnose the signet-ring cell carcinoma without tumor of anal canal. In addition, reconstruction of the perineum for large anal tumors is useful.

[A Case of Robot-Assisted Abdominoperineal Resection with Prostatectomy for Locally Advanced Rectal Cancer].

The patient was referred to our hospital because of bloody stool and anorectal pain, and a colonoscopy revealed a tumor in the lower rectum. Although no distant metastasis was found, the tumor was suspected to have invaded the distal prostate. Neoadjuvant chemoradiotherapy(45 Gy/25 Fr with S-1)resulted in tumor shrinkage and symptomatic improvement, however, the primary tumor remained in close proximity to the prostate and urethra. Thus, we performed a robot-assisted abdominoperineal resection and Retzius-sparing prostatectomy in collaboration with the urology department. The surgical margins were negative and radical resection was achieved. Although minor vesicourethral anastomotic leakage was observed, it recovered conservatively. The patient has been alive 1 year postoperatively without recurrence. The patient initially had urinary incontinence, but it gradually improved. Although a total pelvic resection could have been considered, the robot-assisted surgery made it possible to preserve the urinary tract. The future application of robot-assisted surgery in extended surgery is expected.

Wnt5a in cancer-associated fibroblasts promotes colorectal cancer progression.

Cancer-associated fibroblasts (CAFs) are a major component of the tumor microenvironment and have been shown to promote cancer aggressiveness. In our previous study, analysis of expression profiles obtained from paired CAFs and normal fibroblasts from colorectal cancer (CRC) tissue revealed that gene sets related to the Wnt signaling pathway were highly enriched in colorectal CAFs. Furthermore, among the components of the ß-catenin-independent Wnt pathway, Wnt5a was highly expressed in CAFs. Since Wnt5a is considered to be a regulator of CRC progression in CAFs, we performed immunohistochemical analysis on Wnt5a in 171 patients who underwent surgery for CRC. Positive staining for Wnt5a was often found in cancer stroma, particularly in fibromatous areas, although the immunoreactivity for Wnt5a was weak in cancer cells. Wnt5a status in CAFs was significantly associated with tumor size, depth of invasion, lymphatic and vascular invasion, lymph node metastasis, TNM stage, and recurrence. Subsequent in vitro analyses using human recombinant Wnt5a protein revealed that cancer cell proliferation and migration were significantly increased by stimulation with Wnt5a. Our findings suggest that Wnt5a-derived CAFs play a crucial role in CRC progression and have potential as a target of anti-cancer therapies.

The association between ERK inhibitor sensitivity and molecular characteristics in colorectal cancer.

The mitogen-activated protein kinase (MAPK) pathway plays an important role in the colorectal cancer (CRC) progression, being supposed to be activated by the gene mutations, such as BRAF or KRAS. Although the inhibitors of extracellular signal-regulated kinase (ERK) have demonstrated efficacy in the cells with the BRAF or KRAS mutations, a clinical response is not always associated with the molecular signature. The patient-derived organoids (PDO) have emerged as a powerful in vitro model system to study cancer, and it has been widely applied for the drug screening. The present study aims to analyze the association between the molecular characteristics which analyzed by next-generation sequencing (NGS) and sensitivity to the ERK inhibitor (i.e., SCH772984) in PDO derived from CRC specimens. A drug sensitivity test for the SCH772984 was conducted using 14 CRC cell lines, and the results demonstrated that the sensitivity was in agreement with the BRAF mutation, but was not completely consistent with the KRAS status. In the drug sensitivity test for PDO, 6 out of 7 cases with either BRAF or KRAS mutations showed sensitivity to the SCH772984, while 5 out of 6 cases of both BRAF and KRAS wild-types were resistant. The results of this study suggested that the molecular status of the clinical specimens are likely to represent the sensitivity in the PDOs but is not necessarily absolutely overlapping. PDO might be able to complement the limitations of the gene panel and have the potential to provide a novel precision medicine.

Resection rate curves by location along the small intestine provide perspectives on characteristics of Crohn's disease.

AIM: Crohn's disease (CD) can affect any part of the gastrointestinal tract; however, the frequency of CD lesions differs by location. This work aimed to examine resection rates by location to clarify locational characteristics of the small intestine in surgical CD cases. METHOD: This was a single-centre retrospective case note review of patients who had undergone resection for CD affecting the small intestine between January 2014 and February 2020. Operative details, including length of the small intestine, location and extent of the resection, identified the pattern of disease. By normalizing these data the resection rate along the length of the intestine was calculated to create resection rate curves. RESULTS: One hundred and twenty six surgical cases were identified. The resection rate curves could be divided into two types: exponential and bimodal. For primary surgery, this depended on whether or not surgery was limited to an ileocolic resection. At subsequent surgery, a previous ileocaecal resection influenced the pattern of disease. The peaks of the bimodal curve were located at the proximal and distal ileum. CONCLUSION: CD patients requiring resection of the small intestine can be divided into terminal ileum type (exponential type) and proximal ileum type (bimodal type). In the future this analytical method may help predict the site of any recurrent disease but also provides a new perspective on the disease.

A Phenylfurocoumarin Derivative Reverses ABCG2-Mediated Multidrug Resistance In Vitro and In Vivo.

The ATP-binding cassette subfamily G member 2 (ABCG2) transporter is involved in the development of multidrug resistance in cancer patients. Many inhibitors of ABCG2 have been reported to enhance the chemosensitivity of cancer cells. However, none of these inhibitors are being used clinically. The aim of this study was to identify novel ABCG2 inhibitors by high-throughput screening of a chemical library. Among the 5812 compounds in the library, 23 compounds were selected in the first screening, using a fluorescent plate reader-based pheophorbide a (PhA) efflux assay. Thereafter, to validate these compounds, a flow cytometry-based PhA efflux assay was performed and 16 compounds were identified as potential inhibitors. A cytotoxic assay was then performed to assess the effect these 16 compounds had on ABCG2-mediated chemosensitivity. We found that the phenylfurocoumarin derivative (R)-9-(3,4-dimethoxyphenyl)-4-((3,3-dimethyloxiran-2-yl)methoxy)-7H-furo [3,2-g]chromen-7-one (PFC) significantly decreased the IC50 of SN-38 in HCT-116/BCRP colon cancer cells. In addition, PFC stimulated ABCG2-mediated ATP hydrolysis, suggesting that this compound interacts with the substrate-binding site of ABCG2. Furthermore, PFC reversed the resistance to irinotecan without causing toxicity in the ABCG2-overexpressing HCT-116/BCRP cell xenograft mouse model. In conclusion, PFC is a novel inhibitor of ABCG2 and has promise as a therapeutic to overcome ABCG2-mediated MDR, to improve the efficiency of cancer chemotherapy.

[A Case of Rectal Cancer with Disseminated Intravascular Coagulation Successfully Treated with Emergent Chemotherapy].

A 70's woman complaining blood stool and lower abdominal pain visited a local doctor and was given the diagnosis of rectal cancer by colonoscopy. CT, MRI, and bone scintigraphy revealed multiple lymph node and bone metastasis and peritoneal dissemination. She had developed disseminated intravascular coagulation(DIC)during hospitalization, and the cause was considered to be disseminated carcinomatosis of the bone marrow. Thus, we emergently started chemotherapy with mFOLFOX6, in conjunction with anticoagulation therapy, and the DIC was resolved 11 days after the introduction. Partial response was achieved and the chemotherapy has been continued after 5 months from the onset of the DIC. Since the prognosis of solid tumor patients who developed DIC has been reported to be extremely poor, prompt introduction of chemotherapy should be considered.

[A Case of Curative Resection after Neoadjuvant Chemotherapy for Locally-Advanced Sigmoid Colon Carcinoma with Urinary Bladder Invasion].

A 56-year-old man was referred to our hospital for multidisciplinary treatment of advanced sigmoid colon carcinoma with a suspected bladder invasion. The patient received 8 courses of modified Leucovorin, fluorouracil, and oxaliplatin (mFOLFOX6)plus panitumumab as neoadjuvant chemotherapy for reliable and safe radical resection after ileostomy construction. There was a significant reduction in the tumor size following chemotherapy; hence, low anterior resection was performed. In addition, since preoperative and intraoperative findings suggested bladder invasion, a total cystectomy with ileal conduit urinary diversion was performed. The pathological diagnosis was ypT4b, N0, M0, and ypStage Ⅱc, with all surgical margins being negative. Subsequently, the patient received adjuvant chemotherapy with 4 courses of mFOLFOX6, and his condition improved with no incidence of cancer recurrence following 8 months after the operation. Neoadjuvant chemotherapy for locally advanced colon cancer is one of the effective treatments for reliable and safe radical resection.

[A Case of Sigmoid Colon Cancer with Thrombotic Microangiopathy].

A 50's man was referred to our hospital because of sigmoid colon cancer with multiple liver metastases and para-aortic lymph node metastases. Although blood test showed elevated D-dimer(50 µg/mL), there was no significant thrombus in contrast-enhanced CT scan. Since cancer pain and symptoms of bowel obstruction had continued after endoscopic stent placement, we resected primary lesion. Despite anemia and elevated D-dimer level had persisted after the operation, there were no obvious bleeding source nor thrombus. Continuous intravenous heparin infusion was started for hypercoagulability. Then, D-dimer and CRP levels were promptly decreased. Since schizocyte and giant platelets were observed in peripheral blood smear, he was eventually diagnosed with thrombotic microangiopathy as a paraneoplastic syndrome. However D- dimer and CRP levels were re-elevated, and it seemed to be necessary to control cancer progression. Thus, cetuximab monotherapy was started considering his performance status. After starting cetuximab, fever and CRP level were immediately improved. Cetuximab appeared to be very effective, but he died of acute subdural hematoma. Continuous intravenous heparin infusion was supposed to be effective in the treatment of thrombotic microangiopathy along with the management of cancer.

[A Case of Radically Resected Sigmoid Colon Cancer with Bladder Invasion after Chemotherapy and Weight Loss in an Extremely Obese Patient].

A 30's extremely obese patient(body mass index: BMI 45 kg/m2)was referred to our hospital with a chief complaint of bloody urine and stool. Colonoscopy revealed a sigmoid colon tumor. Barium enema examination revealed stenosis of the sigmoid colon. CT scan showed a tumor in the sigmoid colon, with bladder invasion. The para-aortic lymph node was partially swollen. We considered surgery to be high risk because of the patient's severe obesity. Therefore, we decided to examine the possibility of radical surgery followed by chemotherapy(mFOLFOX6/cetuximab)with weight reduction. Following this, the tumor had shrunk remarkably, and the patient's BMI decreased from 45 kg/m2 to 39 kg/m2. The visceral fat area was reduced from 298 cm2 to 199 cm2 at the umbilical level. We then performed a sigmoid colectomy with partial resection of the bladder. Thus, chemotherapy combined with weight loss enabled us to perform radical surgery safely for a locally advanced sigmoid colon cancer in a patient with severe obesity.

[Bladder-Sparing Surgery with Preoperative Chemotherapy for Rectal Cancer with Urinary Bladder Involvement-A Case Report].

A 65-year-old male was diagnosed with rectal cancer invading the urinary bladder, swollen para-aorticlymph nodes, and multiple liver metastases in abdominal CT. After 8 courses of mFOLFOX6 plus panitumumab, the rectal cancer, para-aortic lymph nodes metastasis, and liver metastases decreased significantly in size. Rectal cancer and liver metastases were considered resectable, hence low anterior resection of the rectum was performed. Intraoperative frozen section analysis showed negative metastaticinvolvement of the para-aorticlymph nodes and surgical margins of the urinary bladder; therefore, the urinary bladder was completely preserved. Partial resection of the liver was performed 2 months later. In conclusion, the patient showed good surgical and quality of life results. Thus, the bladder-sparing strategy with preoperative chemotherapy could be considered for appropriately selected rectal cancer patients with urinary bladder involvement.

[A Case of Solitary Residual Lateral Pelvic Lymph Node Metastasis Despite Complete Response of Primary Rectal Cancer Followed by Neoadjuvant Chemoradiotherapy].

A 45-year-old woman was referred to our hospital complaining of diarrhea. Colonoscopy showed a rectal tumor. Histological examination showed moderately differentiated adenocarcinoma. A CT scan revealed a tumor extending from the lower rectum to the anal canal with a lateral pelvic lymph node(LPLN)swelling. We administered neoadjuvant chemoradiotherapy (45 Gy/25 Fr, S-1 80mg/m / 2/day)and the tumor and LPLN shrank remarkably, with a clinically complete response by CT and PET-CT. We then performed abdominoperineal resection with D3 lymph node and bilateral LPLN dissection. Pathological examination revealed complete disappearance of the cancer cells in the primary site, while lymph node metastasis was detected in one LPLN. We report here a rare case in which LPLN metastasis remained despite the pathological complete response of the primary tumor.

[A Case of Curatively Resected Locally Recurrent Rectal Mucinous Adenocarcinoma Followed by Preoperative Chemoradiotherapy].

A 60s man was diagnosed with rectal cancer and underwent low anterior resection of the rectum. The pathological diagnosis was mucinous adenocarcinoma, pT3(SS), pN0, pM0, pStage Ⅱ. Two years after the primary surgery, contrast-enhanced CT showed local recurrence on the oral side of the anastomosis. As the tumor had invaded the left seminal vesicle and coccygeus muscle, neoadjuvant chemoradiotherapy(NACRT)(S-1 80mg/m / 2 plus 45 Gy/25 Fr)was performed. After NACRT, abdominoperineal resection, including the left seminal vesicle, coccygeus muscle, and coccygeal bone, was performed. Pathological examination showed a histological response of Grade 2 and that R0 resection was achieved. Although the only radical treatment of locally recurrent rectal cancer is R0 resection, we performed R0 resection with Grade 2 histological response to NACRT.

Gastrointestinal toxicities of 5-fluorouracil increase the proportion of regulatory T cells in intestinal tract: advantages of alternate-day S-1 administration.

BACKGROUND: 5-Fluorouracil (5-FU), a core anticancer agent for malignancies, induces gastrointestinal (GI) toxicities. Despite recent advances in tumor immunology, it still remains unknown how GI toxicities affect antitumor immunity. S-1 is a tegafur-based oral 5-FU prodrug which has been widely introduced in Japan and other countries. The alternate-day S-1 administration has been proposed to minimize its GI and other toxicities without reducing its anticancer efficacy. METHODS: In this study, two S-1 administration regimens were compared in mice to evaluate their impact of GI toxicities on immunity. In the daily group as a standard administration model, S-1 was administered for 14 days on and 14 days off, and in the alternate-day group as a non-GI toxicity model, S-1 was administered every other day for 28 days. As well as physical findings, regulatory T cells, Th1 cells and other cells in murine lymphoid tissues were analyzed with flow cytometry. RESULTS: Only the daily group exhibited body weight loss and GI toxicities. In the daily group, a proportion of regulatory T cells in the intestinal lymphoid tissue were demonstrated to be six-fold higher than in the control without S-1, and the proportion of Th1 cells showed a decreasing trend. However, the alternate-day group exhibited almost no change in T-cell subsets. CONCLUSION: GI toxicities of 5-FU may have a negative influence on antitumor immunity due to increased proportions of regulatory T cells and decreased proportions of Th1 cells. The alternate-day S-1 administration may be a useful regimen with its minimal influence on T-cell subsets.

Usefulness of alternate-day administration of S-1 and leucovorin in a xenograft mouse model of colorectal cancer: a shorter drug-free interval leads to more efficient antitumor effects.

BACKGROUND: A clinical trial of S-1 with leucovorin (S-1/LV) in metastatic colorectal cancer (CRC) patients demonstrated promising efficacy; however, the gastrointestinal toxicities were so severe that it has not been applied in the clinical setting. On the other hand, alternate-day administration of S-1 has been proposed to attenuate the adverse events without reducing its anticancer activity. Our present study was conducted to confirm the feasibility of alternate-day administration of S-1/LV in in vivo xenograft tumor models. METHODS: Mice were treated with S-1/LV in a daily group (2 weeks of administration followed by 2 weeks of withdrawal) or an alternate-day group (administration on alternate days for 4 weeks), then the mice were killed and the xenograft tumors were resected. We compared body weight changes, condition of feces, mucosal injury and myelosuppression and assessed adverse reactions, tumor volume, tumor growth inhibition (TGI) and expression of Ki67, TUNEL, cIAP2 and XIAP to evaluate the antitumor activity and tumor apoptosis. RESULTS: Severe weight loss, diarrhea, mucosal injury and myelosuppression were observed only in the daily group; however, some myelosuppression was also observed in the alternate-day group. The TGI in the alternate-day group was better than in the daily group, possibly resulting from apoptosis due to the suppression of cIAP2 but not XIAP. CONCLUSION: Our findings suggest that alternate-day administration of S-1/LV for CRC treatment can achieve high antitumor activity without severe adverse reactions, and we propose that clinical trials with this regimen should be conducted in CRC patients.

[Two cases of advanced gastric cancer achieved pathological complete response by preoperative chemotherapy with S-1 plus weekly low-dose CDDP].

We report 2 cases of resectable advanced gastric cancer who achieved pathological complete response by preoperative chemotherapy with S-1 plus weekly low-dose cisplatin (CDDP). S-1 (80 mg/m²)was administered consecutively for 21 days followed by 14 days' rest, with CDDP (25mg/m²) injected on days 1, 8, and 21. Case 1: A man in his 70s diagnosed with cStage III gastric cancer with lymph node metastases received 2 courses of preoperative chemotherapy with S-1 plus weekly low-dose CDDP followed by total gastrectomy with D2 lymph node dissection. Case 2: A man in his 60s diagnosed with cStage III gastric cancer with lymph node metastases received 4 courses of preoperative chemotherapy with S-1 plus weekly low-dose CDDP followed by total gastrectomy with D2 lymph-node dissection. In both cases, postoperative pathological examination revealed no cancer cells in the resected stomach and lymph nodes. The therapeutic effect of preoperative chemotherapy was assessed as Grade 3. With this regimen, we accomplished preoperative chemotherapy successfully without inpatient care. This regimen can be a promising option as preoperative chemotherapy for advanced gastric cancer.

Young-onset peri-anorectal leiomyomatosis: report of a case.

A 23-year-old female was referred with constipation that lasted for 2 years. Preoperative examinations revealed multiple submucosal tumors beside the anorectum, along with subcutaneous tumors in the left buttock. The pathological diagnosis was leiomyoma. Low anterior resection of the rectum with regional lymph node dissection, along with the resection of the subcutaneous tumors in the left buttock through the transdermal approach, was performed, since multiple tumor formation indicated a high malignant potential. The tumors were diagnosed as multiple leiomyomas with no malignancy. Disease categories such as intravenous leiomyomatosis, leiomyomatosis peritonealis disseminata, Alport syndrome, and Currarino syndrome have been reported to be associated with leiomyomatosis; however, the current case of "peri-anorectal leiomyomatosis" was not classified into any of these. The patient was monitored with careful checkups, and the postoperative course was satisfactory for over 5 years without any sign of recurrence or metastasis. Although the clinicopathological features of this case are quite rare and no therapeutic guidelines for such a disease have yet been established, radical resection should be considered, and the elucidation of the histogenesis of this disease will help establish future therapeutic guidelines.