Spatial and single-cell colocalisation analysis reveals MDK-mediated immunosuppressive environment with regulatory T cells in colorectal carcinogenesis.

BACKGROUND: Cell-cell interaction factors that facilitate the progression of adenoma to sporadic colorectal cancer (CRC) remain unclear, thereby hindering patient survival. METHODS: We performed spatial transcriptomics on five early CRC cases, which included adenoma and carcinoma, and one advanced CRC. To elucidate cell-cell interactions within the tumour microenvironment (TME), we investigated the colocalisation network at single-cell resolution using a deep generative model for colocalisation analysis, combined with a single-cell transcriptome, and assessed the clinical significance in CRC patients. FINDINGS: CRC cells colocalised with regulatory T cells (Tregs) at the adenoma-carcinoma interface. At early-stage carcinogenesis, cell-cell interaction inference between colocalised adenoma and cancer epithelial cells and Tregs based on the spatial distribution of single cells highlighted midkine (MDK) as a prominent signalling molecule sent from tumour epithelial cells to Tregs. Interaction between MDK-high CRC cells and SPP1+ macrophages and stromal cells proved to be the mechanism underlying immunosuppression in the TME. Additionally, we identified syndecan4 (SDC4) as a receptor for MDK associated with Treg colocalisation. Finally, clinical analysis using CRC datasets indicated that increased MDK/SDC4 levels correlated with poor overall survival in CRC patients. INTERPRETATION: MDK is involved in the immune tolerance shown by Tregs to tumour growth. MDK-mediated formation of the TME could be a potential target for early diagnosis and treatment of CRC. FUNDING: Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Science Research; OITA Cancer Research Foundation; AMED under Grant Number; Japan Science and Technology Agency (JST); Takeda Science Foundation; The Princess Takamatsu Cancer Research Fund.

Implementable assay for monitoring minimum residual disease after radical treatment for colorectal cancer.

Considering the cost and invasiveness of monitoring postoperative minimal residual disease (MRD) of colorectal cancer (CRC) after adjuvant chemoradiotherapy (ACT), we developed a favorable approach based on methylated circulating tumor DNA to detect MRD after radical resection. Analyzing the public database, we identified the methylated promoter regions of the genes FGD5, GPC6, and MSC. Using digital polymerase chain reaction (dPCR), we termed the "amplicon of methylated sites using a specific enzyme" assay as "AMUSE." We examined 180 and 114 pre- and postoperative serial plasma samples from 28 recurrent and 19 recurrence-free pathological stage III CRC patients, respectively. The results showed 22 AMUSE-positive of 28 recurrent patients (sensitivity, 78.6%) and 17 AMUSE-negative of 19 recurrence-free patients (specificity, 89.5%). AMUSE predicted recurrence 208 days before conventional diagnosis using radiological imaging. Regarding ACT evaluation by the reactive response, 19 AMUSE-positive patients during their second or third blood samples showed a significantly poorer prognosis than the other patients (p = 9E-04). The AMUSE assay stratified four groups by the altered patterns of tumor burden postoperatively. Interestingly, only 34.8% of cases tested AMUSE-negative during ACT treatment, indicating eligibility for ACT. The AMUSE assay addresses the clinical need for accurate MRD monitoring with universal applicability, minimal invasiveness, and cost-effectiveness, thereby enabling the timely detection of recurrences. This assay can effectively evaluate the efficacy of ACT in patients with stage III CRC following curative resection. Our study strongly recommends reevaluating the clinical application of ACT using the AMUSE assay.

Paneth-like cells produced from OLFM4+ stem cells support OLFM4+ stem cell growth in advanced colorectal cancer.

Tumor tissues consist of heterogeneous cells that originate from stem cells; however, their cell fate determination program remains incompletely understood. Using patient-derived organoids established from patients with advanced colorectal cancer (CRC), we evaluated the potential of olfactomedin 4 (OLFM4)+ stem cells to produce a bifurcated lineage of progenies with absorptive and secretory properties. In the early phases of organoid reconstruction, OLFM4+ cells preferentially gave rise to secretory cells. Additionally, we found that Paneth-like cells, which do not exist in the normal colon, were induced in response to Notch signaling inhibition. Video recordings of single OLFM4+ cells revealed that organoids containing Paneth-like cells were effectively propagated and that their selective ablation led to organoid collapse. In tumor tissues, Paneth-like cells were identified only in the region where tumor cells lost cell adhesion. These findings indicate that Paneth-like cells are directly produced by OLFM4+ stem cells and that their interaction contributes to tumor formation by providing niche factors. This study reveals the importance of the cell fate specification program for building a complete tumor cellular ecosystem, which might be targeted with novel therapeutics.

BET protein-dependent E2F pathway activity confers bell-shaped type resistance to tankyrase inhibitors in APC-mutated colorectal cancer.

WNT/ß-catenin signaling is aberrantly activated in colorectal cancer (CRC) mainly by loss-of-function mutations in adenomatous polyposis coli (APC) and is involved in tumor progression. Tankyrase inhibitors, which suppress WNT/ß-catenin signaling, are currently in pre-clinical and clinical trials. However, the mechanisms of resistance to tankyrase inhibitors remain unclear. In this study, we established tankyrase inhibitor-resistant CRC cells, JC73-RK100, from APC-mutated patient-derived CRC cells. JC73-RK100 cells and several CRC cell lines were sensitive to tankyrase inhibitors at low concentrations but were resistant at high concentrations, showing an intrinsic/acquired bell-shaped dose response. Mechanistically, tankyrase inhibitors at high concentrations promoted BRD3/4-dependent E2F target gene transcription and over-activated cell cycle progression in these cells. BET inhibitors canceled the bell-shaped dose response to tankyrase inhibitors. Combination of tankyrase and BET inhibitors significantly suppressed tumor growth in a mouse xenograft model. These observations suggest that the combination of tankyrase and BET inhibitors may be a useful therapeutic approach to overcome the resistance of a subset of CRCs to tankyrase inhibitors.

APC/PIK3CA mutations and ß-catenin status predict tankyrase inhibitor sensitivity of patient-derived colorectal cancer cells.

BACKGROUND: Aberrant WNT/ß-catenin signaling drives carcinogenesis. Tankyrases poly(ADP-ribosyl)ate and destabilize AXINs, ß-catenin repressors. Tankyrase inhibitors block WNT/ß-catenin signaling and colorectal cancer (CRC) growth. We previously reported that 'short' APC mutations, lacking all seven ß-catenin-binding 20-amino acid repeats (20-AARs), are potential predictive biomarkers for CRC cell sensitivity to tankyrase inhibitors. Meanwhile, 'Long' APC mutations, which possess more than one 20-AAR, do not predict inhibitor-resistant cells. Thus, additional biomarkers are needed to precisely predict the inhibitor sensitivity. METHODS: Using 47 CRC patient-derived cells (PDCs), we examined correlations between the sensitivity to tankyrase inhibitors (G007-LK and RK-582), driver mutations, and the expressions of signaling factors. NOD.CB17-Prkdcscid/J and BALB/c-nu/nu xenograft mice were treated with RK-582. RESULTS: Short APC mutant CRC cells exhibited high/intermediate sensitivities to tankyrase inhibitors in vitro and in vivo. Active ß-catenin levels correlated with inhibitor sensitivity in both short and long APC mutant PDCs. PIK3CA mutations, but not KRAS/BRAF mutations, were more frequent in inhibitor-resistant PDCs. Some wild-type APC PDCs showed inhibitor sensitivity in a ß-catenin-independent manner. CONCLUSIONS: APC/PIK3CA mutations and ß-catenin predict the sensitivity of APC-mutated CRC PDCs to tankyrase inhibitors. These observations may help inform the strategy of patient selection in future clinical trials of tankyrase inhibitors.

Head-to-head comparison of three chelates reveals DOTAGA promising for 225 Ac labeling of anti-FZD10 antibody OTSA101.

To select the most suitable chelate for 225 Ac radiolabeling of the anti-FZD10 antibody OTSA101, we directly compared three chelates: S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA), 2,2',2″-(10-(1-carboxy-4-((4-isothiocyanatobenzyl)amino)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (p-SCN-Bn-DOTAGA), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DO3A-NHS-ester). We evaluated the binding affinity of the chelate-conjugated OTSA101 antibodies, as well as the labeling efficiency and stability in murine serum of 225 Ac-labeled OTSA101 as in vitro properties. The biodistribution, intratumoral distribution, absorbed doses, and therapeutic effects of the chelate-conjugated OTSA101 antibodies were assessed in the synovial sarcoma mouse model SYO-1. Of the three conjugates, DOTAGA conjugation had the smallest impact on the binding affinity (p < 0.01). The labeling efficiencies of DOTAGA-OTSA101 and DO3A-OTSA101 were 1.8-fold higher than that of DOTA-OTSA101 (p < 0.01). The stabilities were similar between 225 Ac-labeled DOTA-OTSA101, DOTAGA-OTSA101, and DO3A-OTSA101in serum at 37 and 4°C. The dosimetric analysis based on the biodistribution revealed significantly higher tumor-absorbed doses by 225 Ac-labeled DOTA-OTSA101 and DOTAGA-OTSA101 compared with 225 Ac-DO3A-OTSA101 (p < 0.05). 225 Ac-DOTAGA-OTSA101 exhibited the highest tumor-to-bone marrow ratio, with bone marrow being the dose-limiting tissue. The therapeutic and adverse effects were not significantly different between the three conjugates. Our findings indicate that among the three evaluated chelates, DOTAGA appears to be the most promising chelate to produce 225 Ac-labeled OTSA101 with high binding affinity and high radiochemical yields while providing high absorbed doses to tumors and limited absorbed doses to bone marrow.

Prognostic factors of para-aortic lymph node metastasis from colorectal cancer in highly selected patients undergoing para-aortic lymph node dissection.

PURPOSE: We investigated the surgical outcomes of para-aortic lymph node (PALN) dissection in patients with colorectal cancer and assessed the prognostic factors related to the survival. METHODS: This single-center retrospective study included 31 patients with synchronous or metachronous PALN metastasis from colorectal cancer who underwent PALN dissection between January 2006 and December 2018. RESULTS: Twenty-one patients had synchronous PALN metastasis, and 10 had metachronous PALN metastasis. Seven patients had either simultaneous distant metastasis or a history of distant metastasis other than PALN metastasis at the time of PALN dissection. Eighteen patients underwent adjuvant chemotherapy. The 5-year overall and recurrence-free survival rates were 54.2 and 17.2%, respectively. A multivariable analysis revealed that rectal cancer, metachronous PALN metastasis, and three or more pathological PALN metastases were significantly poor prognostic factors for the recurrence-free survival. Among patients with rectal cancer, lower rectal cancer and lateral pelvic lymph node metastasis were poor prognostic factors for the overall survival. CONCLUSION: Curative PALN dissection for PALN metastasis from colorectal cancer is feasible with favorable long-term outcomes. A multidisciplinary approach, including surgery and chemotherapy, is needed for colorectal cancer with PALN metastasis to improve the long-term outcomes.

Subclonal accumulation of immune escape mechanisms in microsatellite instability-high colorectal cancers.

BACKGROUND: Intratumor heterogeneity (ITH) in microsatellite instability-high (MSI-H) colorectal cancer (CRC) has been poorly studied. We aimed to clarify how the ITH of MSI-H CRCs is generated in cancer evolution and how immune selective pressure affects ITH. METHODS: We reanalyzed public whole-exome sequencing data on 246 MSI-H CRCs. In addition, we performed a multi-region analysis from 6 MSI-H CRCs. To verify the process of subclonal immune escape accumulation, a novel computational model of cancer evolution under immune pressure was developed. RESULTS: Our analysis presented the enrichment of functional genomic alterations in antigen-presentation machinery (APM). Associative analysis of neoantigens indicated the generation of immune escape mechanisms via HLA alterations. Multiregion analysis revealed the clonal acquisition of driver mutations and subclonal accumulation of APM defects in MSI-H CRCs. Examination of variant allele frequencies demonstrated that subclonal mutations tend to be subjected to selective sweep. Computational simulations of tumour progression with the interaction of immune cells successfully verified the subclonal accumulation of immune escape mutations and suggested the efficacy of early initiation of an immune checkpoint inhibitor (ICI) -based treatment. CONCLUSIONS: Our results demonstrate the heterogeneous acquisition of immune escape mechanisms in MSI-H CRCs by Darwinian selection, providing novel insights into ICI-based treatment strategies.

Spatiotemporal commonality of the TCR repertoire in a T-cell memory murine model and in metastatic human colorectal cancer.

Immune checkpoint inhibitors (ICIs) have shown superior clinical responses and significantly prolong overall survival (OS) for many types of cancer. However, some patients exhibit long-term OS, whereas others do not respond to ICI therapy at all. To develop more effective and long-lasting ICI therapy, understanding the host immune response to tumors and the development of biomarkers are imperative. In this study, we established an MC38 immunological memory mouse model by administering an anti-PD-L1 antibody and evaluating the detailed characteristics of the immune microenvironment including the T cell receptor (TCR) repertoire. In addition, we found that the memory mouse can be established by surgical resection of residual tumor following anti-PD-L1 antibody treatment with a success rate of > 40%. In this model, specific depletion of CD8 T cells revealed that they were responsible for the rejection of reinoculated MC38 cells. Analysis of the tumor microenvironment (TME) of memory mice using RNA-seq and flow cytometry revealed that memory mice had a quick and robust immune response to MC38 cells compared with naïve mice. A TCR repertoire analysis indicated that T cells with a specific TCR repertoire were expanded in the TME, systemically distributed, and preserved in the host for a long time period. We also identified shared TCR clonotypes between serially resected tumors in patients with colorectal cancer (CRC). Our results suggest that memory T cells are widely preserved in patients with CRC, and the MC38 memory model is potentially useful for the analysis of systemic memory T-cell behavior.

Identification of T Cell Receptors Targeting a Neoantigen Derived from Recurrently Mutated FGFR3.

Immunotherapies, including immune checkpoint blockades, play a critically important role in cancer treatments. For immunotherapies, neoantigens, which are generated by somatic mutations in cancer cells, are thought to be good targets due to their tumor specificity. Because neoantigens are unique in individual cancers, it is challenging to develop personalized immunotherapy targeting neoantigens. In this study, we screened "shared neoantigens", which are specific types of neoantigens derived from mutations observed commonly in a subset of cancer patients. Using exome sequencing data in the Cancer Genome Atlas (TCGA), we predicted shared neoantigen peptides and performed in vitro screening of shared neoantigen-reactive CD8+ T cells using peripheral blood from healthy donors. We examined the functional activity of neoantigen-specific T cell receptors (TCRs) by generating TCR-engineered T cells. Among the predicted shared neoantigens from TCGA data, we found that the mutated FGFR3Y373C peptide induced antigen-specific CD8+ T cells from the donor with HLA-A*02:06 via an ELISPOT assay. Subsequently, we obtained FGFR3Y373C-specific CD8+ T cell clones and identified two different sets of TCRs specifically reactive to FGFR3Y373C. We found that the TCR-engineered T cells expressing FGFR3Y373C-specific TCRs recognized the mutated FGFR3Y373C peptide but not the corresponding wild-type peptide. These two FGFR3Y373C-specific TCR-engineered T cells showed cytotoxic activity against mutated FGFR3Y373C-loaded cells. These results imply the possibility of strategies of immunotherapies targeting shared neoantigens, including cancer vaccines and TCR-engineered T cell therapies.

Spatial and single-cell transcriptomics decipher the cellular environment containing HLA-G+ cancer cells and SPP1+ macrophages in colorectal cancer.

The cellular interactions in the tumor microenvironment of colorectal cancer (CRC) are poorly understood, hindering patient treatment. In the current study, we investigate whether events occurring at the invasion front are of particular importance for CRC treatment strategies. To this end, we analyze CRC tissues by combining spatial transcriptomics from patients with a public single-cell transcriptomic atlas to determine cell-cell interactions at the invasion front. We show that CRC cells are localized specifically at the invasion front. These cells induce human leukocyte antigen G (HLA-G) to produce secreted phosphoprotein 1 (SPP1)+ macrophages while conferring CRC cells with anti-tumor immunity, as well as proliferative and invasive properties. Taken together, these findings highlight the signaling between CRC cell populations and stromal cell populations at the cellular level.

Mutated genes on ctDNA detecting postoperative recurrence presented reduced neoantigens in primary tumors in colorectal cancer cases.

The detection and sequencing of the mutated ctDNA is one of the irreplaceable clinical measures in the postoperative management of colorectal cancer (CRC) cases. However, we are curious to comprehend the essential traits of mutated genes comprising metastatic sites out of whole mutated genes in primary sites. In the current retrospective study, we conducted target resequencing of ctDNA using 47 plasma samples and established a cancer panel carrying the commonly mutated genes between primary and recurrent tumors. We found that mutated genes in ctDNA indicated immune-resistance traits with respect to the impaired ability to present neoantigens by loss of expression or binding affinity to HLA in the primary tumor. Compared with the estimated neoantigens from all mutated genes in primary tumors, the neoantigen peptides from commonly mutated genes on the panel showed abundant expression but no binding affinity to HLA. Therefore, ctDNA mutations can be frequently and postoperatively detected to identify recurrence; however, these mutated genes were derived from immune-tolerated clones owing to the loss of neoantigen presentation in primary CRC tumors.

A large-scale targeted proteomics of plasma extracellular vesicles shows utility for prognosis prediction subtyping in colorectal cancer.

PURPOSE: The pathogenesis of cancers depends on the molecular background of each individual patient. Therefore, verifying as many biomarkers as possible and clarifying their relationships with each disease status would be very valuable. We performed a large-scale targeted proteomics analysis of plasma extracellular vesicles (EVs) that may affect tumor progression and/or therapeutic resistance. EXPERIMENTAL DESIGN: Plasma EVs from 59 were collected patients with colorectal cancer (CRC) and 59 healthy controls (HC) in cohort 1, and 150 patients with CRC in cohort 2 for the large-scale targeted proteomics analysis of 457 proteins as candidate CRC markers. The Mann-Whitney-Wilcoxon test and random forest model were applied in cohort 1 to select promising markers. Consensus clustering was applied to classify patients with CRC in cohort 2. The Kaplan-Meier method and Cox regression analysis were performed to identify potential molecular factors contributing to the overall survival (OS) of patients. RESULTS: In the analysis of cohort 1, 99 proteins were associated with CRC. The analysis of cohort 2 revealed two clusters showing significant differences in OS (p = 0.017). Twelve proteins, including alpha-1-acid glycoprotein 1 (ORM1), were suggested to be associated with the identified CRC subtypes, and ORM1 was shown to significantly contribute to OS, suggesting that ORM1 might be one of the factors closely related to the OS. CONCLUSIONS: The study identified two novel subtypes of CRC, which exhibit differences in OS, as well as important biomarker proteins that are closely related to the identified subtypes. Liquid biopsy assessment with targeted proteomics analysis was proposed to be crucial for predicting the CRC prognosis.

RNaseH2A downregulation drives inflammatory gene expression via genomic DNA fragmentation in senescent and cancer cells.

Cellular senescence caused by oncogenic stimuli is associated with the development of various age-related pathologies through the senescence-associated secretory phenotype (SASP). SASP is mediated by the activation of cytoplasmic nucleic acid sensors. However, the molecular mechanism underlying the accumulation of nucleotide ligands in senescent cells is unclear. In this study, we revealed that the expression of RNaseH2A, which removes ribonucleoside monophosphates (rNMPs) from the genome, is regulated by E2F transcription factors, and it decreases during cellular senescence. Residual rNMPs cause genomic DNA fragmentation and aberrant activation of cytoplasmic nucleic acid sensors, thereby provoking subsequent SASP factor gene expression in senescent cells. In addition, RNaseH2A expression was significantly decreased in aged mouse tissues and cells from individuals with Werner syndrome. Furthermore, RNaseH2A degradation using the auxin-inducible degron system induced the accumulation of nucleotide ligands and induction of certain tumourigenic SASP-like factors, promoting the metastatic properties of colorectal cancer cells. Our results indicate that RNaseH2A downregulation provokes SASP through nucleotide ligand accumulation, which likely contributes to the pathological features of senescent, progeroid, and cancer cells.

[A Case of Perforation of Unresectable Esophageal Cancer during Nivolumab Therapy].

A 46-year-old man visited our hospital complaining of dysphagia. He was diagnosed with unresectable esophageal cancer with multiple lung metastases(cStage Ⅳb)and gastric cancer(L, Gre, T3N+M0, cStage Ⅲ). The esophageal lesion and the lung metastatic lesions showed shrinkage initially with 5-FU, CDDP(FP)therapy but then re-grew; therefore, the therapy was changed to nivolumab therapy. After three courses of nivolumab therapy, the patient visited our hospital with a high fever. He was admitted as an emergency patient with a diagnosis of esophageal perforation and mediastinal abscess. CT- guided drainage was performed, and a self-expanding metal stent(SEMS)was placed. He was discharged on the 31st day of hospitalization and nivolumab therapy was resumed. We report the first case of esophageal perforation during immunotherapy with nivolumab therapy for esophageal cancer.

Differential ion mobility mass spectrometry in immunopeptidomics identifies neoantigens carrying colorectal cancer driver mutations.

Understanding the properties of human leukocyte antigen (HLA) peptides (immunopeptides) is essential for precision cancer medicine, while the direct identification of immunopeptides from small biopsies of clinical tissues by mass spectrometry (MS) is still confronted with technical challenges. Here, to overcome these hindrances, high-field asymmetric waveform ion mobility spectrometry (FAIMS) is introduced to conduct differential ion mobility (DIM)-MS by seamless gas-phase fractionation optimal for scarce samples. By established DIM-MS for immunopeptidomics analysis, on average, 42.9 mg of normal and tumor colorectal tissues from identical patients (n = 17) were analyzed, and on average 4921 immunopeptides were identified. Among these 44,815 unique immunopeptides, two neoantigens, KRAS-G12V and CPPED1-R228Q, were identified. These neoantigens were confirmed by synthetic peptides through targeted MS in parallel reaction monitoring (PRM) mode. Comparison of the tissue-based personal immunopeptidome revealed tumor-specific processing of immunopeptides. Since the direct identification of neoantigens from tumor tissues suggested that more potential neoantigens have yet to be identified, we screened cell lines with known oncogenic KRAS mutations and identified 2 more neoantigens that carry KRAS-G12V. These results indicated that the established FAIMS-assisted DIM-MS is effective in the identification of immunopeptides and potential recurrent neoantigens directly from scarce samples such as clinical tissues.

A case of acute appendicitis incarcerated in obturator hernia.

An 85 year-old woman was transferred with a chief complaint of right thigh pain persisting for 5 days. Abdominal contrast-enhanced computed tomography clearly depicted a swollen appendix incarcerated in the right obturator cavity. She underwent an emergent laparoscopic appendectomy and the simultaneous repair of the obturator hernia. At laparoscopy, appendix was found to be incarcerated in the right obturator canal. The incarcerated appendix was successfully flushed out from the sac by spurting saline into the obturator hernia sac through the catheter inserted into the hernia sac. After a laparoscopic appendectomy, the hernia orifice was repaired using the uterine flap. The patient was discharged from the hospital without any sequelae. This report demonstrates a very rare case of obturator hernia incarcerated with appendix. Although patients with obturator hernia incarcerated with small intestine present with the symptoms related to bowel obstruction, patients with incarceration of appendix do not. Moreover, they would show no typical abdominal symptoms associated with acute appendicitis. Therefore, it is important to perform a radiological evaluation promptly to make a definitive diagnosis when a patient with persisting pain of the right thigh or right ileac fossa with a possibility of obturator hernia with incarceration of the appendix is encountered.

Acquired resistance to BRAF inhibitors is mediated by BRAF splicing variants in BRAF V600E mutation-positive colorectal neuroendocrine carcinoma.

Neuroendocrine carcinomas (NECs), a poorly differentiated subtype of neuroendocrine neoplasms, are aggressive and have a poor prognosis. Colorectal neuroendocrine carcinomas (CRC-NECs) are observed in about 0.6% of all patients with CRC. Interestingly, patients with CRC-NECs show higher frequencies of BRAF mutation than typical CRC. BRAF V600E mutation-positive CRC-NECs were shown to be sensitive to BRAF inhibitors and now are treated by BRAF inhibitors. Similar to the other BRAF V600E mutated cancers, resistances against BRAF inhibitors have been observed, but the resistance mechanisms are still unclear. In this study, we established BRAF V600E mutated CRC-NEC cell line directly from surgical specimens and experimentally obtained BRAF inhibitor dabrafenib resistant cell lines. The resistant cells are revealed to express at least three types of BRAF splicing variants harboring V600E-mutation, and contribute to RAF/MEK/ERK pathway activation. In these cells, MEK and ERK inhibitors but not dabrafenib significantly suppressed cell growth and survival. Thus, in BRAF V600E mutation-positive CRC-NECs, BRAF splicing variants activate the RAF/MEK/ERK pathway and contribute to acquire BRAF inhibitor resistance. Hence, MEK or ERK are potential therapeutic targets to overcome BRAF resistance.

Integration of human inspection and artificial intelligence-based morphological typing of patient-derived organoids reveals interpatient heterogeneity of colorectal cancer.

Colorectal cancer (CRC) is a heterogenous disease, and patients have differences in therapeutic response. However, the mechanisms underlying interpatient heterogeneity in the response to chemotherapeutic agents remain to be elucidated, and molecular tumor characteristics are required to select patients for specific therapies. Patient-derived organoids (PDOs) established from CRCs recapitulate various biological characteristics of tumor tissues, including cellular heterogeneity and the response to chemotherapy. Patient-derived organoids established from CRCs show various morphologies, but there are no criteria for defining these morphologies, which hampers the analysis of their biological significance. Here, we developed an artificial intelligence (AI)-based classifier to categorize PDOs based on microscopic images according to their similarity in appearance and classified tubular adenocarcinoma-derived PDOs into six types. Transcriptome analysis identified differential expression of genes related to cell adhesion in some of the morphological types. Genes involved in ribosome biogenesis were also differentially expressed and were most highly expressed in morphological types showing CRC stem cell properties. We identified an RNA polymerase I inhibitor, CX-5641, to be an upstream regulator of these type-specific gene sets. Notably, PDO types with increased expression of genes involved in ribosome biogenesis were resistant to CX-5461 treatment. Taken together, these results uncover the biological significance of the morphology of PDOs and provide novel indicators by which to categorize CRCs. Therefore, the AI-based classifier is a useful tool to support PDO-based cancer research.

Lymphocytes in tumor-draining lymph nodes co-cultured with autologous tumor cells for adoptive cell therapy.

BACKGROUND: Tumor-draining lymph nodes (TDLNs) are primary sites, where anti-tumor lymphocytes are primed to tumor-specific antigens and play pivotal roles in immune responses against tumors. Although adoptive cell therapy (ACT) using lymphocytes isolated from TDLNs were reported, characterization of immune activity of lymphocytes in TDLNs to tumor cells was not comprehensively performed. Here, we demonstrate TDLNs to have very high potential as cell sources for immunotherapy. METHODS: Lymphocytes from TDLNs resected during surgical operation were cultured with autologous-tumor cells for 2 weeks and evaluated tumor-reactivity by IFNγ ELISPOT assay. We investigated the commonality of T cell receptor (TCR) clonotypes expanded by the co-culture with tumor cells with those of tumor infiltrating lymphocytes (TILs). RESULTS: We found that that TCR clonotypes of PD-1-expressing CD8+ T cells in lymph nodes commonly shared with those of TILs in primary tumors and lymphocytes having tumor-reactivity and TCR clonotypes shared with TILs could be induced from non-metastatic lymph nodes when they were co-cultured with autologous tumor cells. CONCLUSION: Our results imply that tumor-reactive effector T cells were present even in pathologically non-metastatic lymph nodes and could be expanded in vitro in the presence of autologous tumor cells and possibly be applied for ACT.