Drug screening by uniform patient derived colorectal cancer hydro-organoids.

As the importance of organoids increases, the need to develop organoid culture systems suitable for basic biological and clinical applications is being emphasized. However, there is still an unmet need to produce functionally complex and scalable uniform organoids. Here, we demonstrate a scalable organoid production platform with 8 well strips and a total of 8 × 9 microwells per strip using organoids derived from colorectal cancer tissue. The new culture platform is a format in which single cells are self-organized into organoids in culture medium supplemented with 2% Matrigel. It is functionally compatible with existing 96 well plates and Matrigel based conventional organoid culture methods. The consistency, uniformity and reproducibility of organoid produced on the new platform have been significantly improved compared to those of conventional plates. Importantly, Hydro-organoids are functionally identical to conventional Matrigel organoids, but show better consistency in drug screening. Our results show the possibility that Hydro-organoids can be used in high-throughput assays and incorporated into drug screening models to predict clinical outcomes.

Significance of Radial Margin in Patients Undergoing Complete Mesocolic Excision for Colon Cancer.

BACKGROUND: Comparable to circumferential resection margin in rectal cancer, radial margin is a potential prognostic factor in colon cancer that has just begun to be studied. No previous studies have investigated the influence of radial margin in the context of complete mesocolic excision. OBJECTIVE: This study aimed to examine the impact of radial margin on oncologic outcomes after complete mesocolic excision for colon cancer. DESIGN: We retrospectively reviewed patients with stage I to III colon cancer who underwent curative resection from October 2010 to March 2013. SETTINGS: This study was conducted using the prospective colorectal cancer registry of Severance hospital. PATIENTS: A total of 834 consecutive patients who underwent complete mesocolic excision for colon adenocarcinoma were included. INTERVENTIONS: We assigned patients into 3 groups according to radial margin distance: group A, radial margin ≥2.0 mm; group B, 1.0 ≤ radial margin < 2.0 mm; group C, radial margin <1 mm. MAIN OUTCOMES AND MEASURES: Overall survival and disease-free survival were estimated. RESULTS: On adjusted Cox regression analysis, only group C was predictive of reduced overall survival (HR, 1.90; 95% CI, 1.11-3.25; p = 0.018) and disease-free survival (HR, 1.93; 95% CI, 1.28-2.89; p = 0.001). We thereby defined radial margin threatening as radial margin <1 mm. Postoperative 5-fluorouracil (HR, 0.86; 95% CI, 0.35-2.10; p = 0.743) and FOLFOX (HR, 1.23; 95% CI, 0.57-2.64; p = 0.581) chemotherapy did not affect disease-free survival in patients with radial margin threatening. LIMITATIONS: This study has the limitations inherent in all retrospective, single-institution studies. CONCLUSIONS: Even with complete mesocolic excision, radial margin <1 mm was an independent predictor of survival and recurrence. This finding suggests that special efforts for obtaining a clear radial margin may be necessary in locally advanced colon cancer. See Video Abstract at http://links.lww.com/DCR/B125. IMPORTANCIA DEL MARGEN RADIAL EN PACIENTES SOMETIDOS A ESCISIÓN MESOCÓLICA COMPLETA PARA CÁNCER DEL COLON: Comparable al margen de resección circunferencial en cáncer rectal, el margen radial en cáncer de colon, es un factor pronóstico potencial, que recientemente comienza a estudiarse. Ningún estudio previo ha investigado la influencia del margen radial, en el contexto de la escisión mesocólica completa.Examinar en cáncer de colon, el impacto del margen radial en los resultados oncológicos, después de la escisión mesocólica completa.Revisión retrospectiva de pacientes con cáncer de colon en estadio I-III, sometidos a resección curativa de octubre 2010 a marzo 2013.Este estudio se realizó utilizando un registro prospectivo de cáncer colorrectal del hospital Severance.Se incluyeron un total de 834 pacientes consecutivos con adenocarcinoma de colon, sometidos a escisión mesocólica completa. Dividimos a los pacientes en 3 grupos según la distancia del margen radial: grupo A, margen radial ≥ 2.0 mm; grupo B, 1.0 ≤ margen radial <2.0 mm; grupo C, margen radial <1 mm.Se estimó la supervivencia general y la supervivencia libre de enfermedad.En el análisis de regresión de Cox ajustado, solo el grupo C fue predictivo de supervivencia global reducida (HR, 1.90; IC 95%, 1.11-3.25; p = 0.018) y supervivencia libre de enfermedad (HR, 1.93; IC 95%, 1.28-2.89; p = 0.001). Definimos como margen radial amenazante, un margen radial <1 mm. La quimioterapia posoperatoria con 5-FU (HR, 0,86; IC 95%, 0,35-2,10; p = 0.743) y FOLFOX (HR, 1,23; IC 95%, 0,57-2,64; p = 0,581), no afectó la supervivencia libre de enfermedad en pacientes con riesgo de margen radial.Este estudio tiene limitaciones inherentes a todos los estudios retrospectivos de una sola institución.Aun con la escisión mesocólica completa, el margen radial <1 mm fue un predictor independiente de supervivencia y recurrencia. Este hallazgo sugiere que pueden ser necesarios esfuerzos especiales para obtener un claro margen radial, en cáncer de colon localmente avanzado. Consulte Video Resumen en http://links.lww.com/DCR/B125.

A novel long noncoding RNA Linc-ASEN represses cellular senescence through multileveled reduction of p21 expression.

Long noncoding RNAs (lncRNAs) regulating diverse cellular processes implicate in many diseases. However, the function of lncRNAs in cellular senescence remains largely unknown. Here we identify a novel long intergenic noncoding RNA Linc-ASEN expresses in prematurely senescent cells. We find that Linc-ASEN associates with UPF1 by RNA pulldown mass spectrometry analysis, and represses cellular senescence by reducing p21 production transcriptionally and posttranscriptionally. Mechanistically, the Linc-ASEN-UPF1 complex suppressed p21 transcription by recruiting Polycomb Repressive Complex 1 (PRC1) and PRC2 to the p21 locus, and thereby preventing binding of the transcriptional activator p53 on the p21 promoter through histone modification. In addition, the Linc-ASEN-UPF1 complex repressed p21 expression posttranscriptionally by enhancing p21 mRNA decay in association with DCP1A. Accordingly, Linc-ASEN levels were found to correlate inversely with p21 mRNA levels in tumors from patient-derived mouse xenograft, in various human cancer tissues, and in aged mice tissues. Our results reveal that Linc-ASEN prevents cellular senescence by reducing the transcription and stability of p21 mRNA in concert with UPF1, and suggest that Linc-ASEN might be a potential therapeutic target in processes influenced by senescence, including cancer.

VEGF-A drives TOX-dependent T cell exhaustion in anti-PD-1-resistant microsatellite stable colorectal cancers.

Although immune checkpoint blockade therapies have demonstrated clinical efficacy in cancer treatment, harnessing this strategy is largely encumbered by resistance in multiple cancer settings. Here, we show that tumor-infiltrating T cells are severely exhausted in the microsatellite stable (MSS) colorectal cancer (CRC), a representative example of PD-1 blockade-resistant tumors. In MSS CRC, we found wound healing signature to be up-regulated and that T cell exhaustion is driven by vascular endothelial growth factor-A (VEGF-A). We report that VEGF-A induces the expression of transcription factor TOX in T cells to drive exhaustion-specific transcription program in T cells. Using a combination of in vitro, ex vivo, and in vivo mouse studies, we demonstrate that combined blockade of PD-1 and VEGF-A restores the antitumor functions of T cells, resulting in better control of MSS CRC tumors.

Clinicopathological and biomolecular characteristics of stage IIB/IIC and stage IIIA colon cancer: Insight into the survival paradox.

BACKGROUND: A survival paradox of stage IIB/IIC and IIIA colon cancer has been consistently observed throughout revisions of the TNM system. This study aimed to understand this paradox with clinicopathological and molecular differences. METHODS: Clinicopathological characteristics of patients with pathologically confirmed stage IIB/IIC or IIIA colon cancer were retrospectively reviewed from a database. Publicly available molecular data were retrieved, and intrinsic subtypes were identified and subjected to gene sets enrichment analysis (GSEA). RESULTS: Among the 159 patients included in the clinicopathological analysis, those at stage IIB/IIC had worse 3-year disease-free and overall survival than those at stage IIIA (59.3% vs 91.7%, P < 0.001 and 82.7% vs 98.5%, P < 0.001, respectively), even after adjusting for confounding factors. Data of 95 patients were retrieved from public databases, demonstrating a higher frequency of the microsatellite instable subtype in stage IIB/IIC. The consensus molecular subtype distribution pattern differed between the groups. The GSEA further suggested the protumor inflammatory reaction might be more prominent in stage IIB/IIC. CONCLUSIONS: The survival paradox in colon cancer was confirmed and appears to be a multifactorial phenomenon not attributed to a single clinicopathologic factor. However, the greater molecular heterogeneity in stage IIB/IIC could contribute to the poor prognosis.

Impact of Adjuvant Chemotherapy Completion on Oncologic Outcomes in ypTNMstage 2 Rectal Cancer Patients.

PURPOSE: Adjuvant chemotherapy (aCT) in rectal cancer patients who have undergone curative resection after neoadjuvant chemoradiation (nCRT) is controversial. We aimed to investigate the benefits of using aCT and the clinical impact of completing aCT in ypstage 2 rectal cancer patients. METHODS: We retrospectively reviewed clinicopathological data from patients who had undergone radical resection after nCRT between January 2006 and December 2012. In total, 152 patients with ypT3/4N0M0 rectal cancer were included. Of these patients, 139 initiated aCT, while 13 did not receive aCT (no-aCT). Among those who received aCT, 132 patients completed their planned cycles (aCT-completion) whereas 7 did not (aCT-incompletion). All patients received longcourse chemoradiation; a 5-fluorouracil-based regimen was used for nCRT in most patients. The prognostic factors affecting disease-free survival (DFS) and overall survival (OS) were analyzed. RESULTS: The median follow-up duration was 41 months. Demographic data did not differ significantly among the 3 groups. In multivariate analysis, open surgery, a tumor size >2 cm, retrieval of <12 lymph nodes, circumferential resection margin (CRM) positivity and aCT incompletion were independent prognostic factors for poor DFS. Old age (≥60 years), open surgery, CRM positivity, aCT incompletion, and lack of aCT initiation compared to aCT completion were independent prognostic factors for poor OS. CONCLUSION: In ypstage 2 rectal cancer patients, aCT after nCRT and total mesorectal excision affected both DFS and OS; however, only patients who completed planned aCT exhibited survival benefits. Therefore, improving patients' compliance with the completion of aCT is desirable.

Temporal changes in immune cell composition and cytokines in response to chemoradiation in rectal cancer.

We measured systemic changes in the immune response in 92 patients receiving preoperative chemoradiation therapy (CRT) and subsequent surgery for rectal cancer. The peripheral blood was sampled five times from the onset of CRT until surgery. Lymphocytes decreased continuously during CRT but increased after CRT. The increased lymphocyte population was predominantly CD8+ T lymphocytes, which accounted for a significantly larger proportion in patients without residual lymph node metastasis than in those with residual lymph node metastasis. Neutrophils and monocytes decreased during the initial two weeks of CRT but were maintained or increased afterwards. Neutrophil and monocyte counts were significantly lower in patients with a pCR (pathologic complete response) than in those without a pCR two weeks after CRT began but not at the initiation of CRT. All cytokines showed dramatic changes one month after the termination of CRT. Cytokines related to the antitumour immune response increased, and those related to tumour progression decreased. The predictive value of cytokines was not clear. In short, we observed that immune components in peripheral blood are affected by CRT and show dynamic changes over time. We identified biomarker candidates to predict the pathologic response in the future.

Evaluation of recombinant adenovirus-mediated gene delivery for expression of tracer genes in catecholaminergic neurons.

Selective labeling of small populations of neurons of a given phenotype for conventional neuronal tracing is difficult because tracers can be taken up by all neurons at the injection site, resulting in nonspecific labeling of unrelated pathways. To overcome these problems, genetic approaches have been developed that introduce tracer proteins as transgenes under the control of cell-type-specific promoter elements for visualization of specific neuronal pathways. The aim of this study was to explore the use of tracer gene expression for neuroanatomical tracing to chart the complex interconnections of the central nervous system. Genetic tracing methods allow for expression of tracer molecules using cell-type-specific promoters to facilitate neuronal tracing. In this study, the rat tyrosine hydroxylase (TH) promoter and an adenoviral delivery system were used to express tracers specifically in dopaminergic and noradrenergic neurons. Region-specific expression of the transgenes was then analyzed. Initially, we characterized cell-type-specific expression of GFP or RFP in cultured cell lines. We then injected an adenovirus carrying the tracer transgene into several brain regions using a stereotaxic apparatus. Three days after injection, strong GFP expression was observed in the injected site of the brain. RFP and WGA were expressed in a cell-type-specific manner in the cerebellum, locus coeruleus, and ventral tegmental regions. Our results demonstrate that selective tracing of catecholaminergic neuronal circuits is possible in the rat brain using the TH promoter and adenoviral expression.