Kenoki Ohuchida’s research while affiliated with Kyushu University and other places

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Publications (310)


Identification of KPC mouse–derived primary cultures of PDAC inducing infiltration of CD8⁺ T-cells in tumors. (A) Representative 3D-culture phase-contrast images of KrasG12D/+; Trp53R172H/+; Pdx-1-Cre (KPC) mouse–derived primary cultures of pancreatic ductal adenocarcinoma (PDAC) with serum or niche factor medium. We classified the morphological features of 3D-cultures based on PDAC differentiation grade. (B) Schematic of the study design. TIME: tumor immune microenvironment; RNA-seq: RNA sequencing. (C) Primary cultures of PDAC cells (2.0 × 10⁵) were transplanted orthotopically, and tumors were resected three weeks later. CD8α immunohistochemistry (IHC) staining images of mouse PDAC tumors (left) and the number of CD8⁺ T-cells (CD8α-positive cells) per field (right) for each primary culture. Scale bars, 100 μm
RNA sequencing identified specific genes inducing cold TIME in KPC mouse–derived primary cultures of PDAC. (A) Volcano plot of cold and hot TIME groups. Red: hot TIME differentially expressed genes (DEGs), blue: cold TIME DEGs (p < 0.05). Cold tumor induction–related genes are labeled. (B) The expression levels of cold tumor induction–related genes in each KPC mouse–derived primary culture of PDAC
“Cold tumor induction–related genes” and PCDH7 were associated with cold TIME in human PDAC. (A–C, E and F) Single-cell RNA sequencing (scRNA-seq) analysis of a public database of human PDAC patients. (A) Uniform Manifold Approximation and Projection (UMAP) plot showing the clustering of human PDAC. (B) UMAP plot showing the re-clustering of PTPRC (CD45)-positive immune cells. (C) The correlation between “cold tumor induction–related genes” expression and the number of CD8-positive cells per patient. (D) Kaplan–Meier disease-free survival analysis of cold tumor induction–related genes from The Cancer Genome Atlas (TCGA) pancreatic adenocarcinoma samples (n = 89 in each group). (E and F) The correlation between PCDH7 expression and the percentage of CD8-positive cells (E) and Tregs (F) per patient. (G and H) Using TCGA data of human pancreatic adenocarcinoma patients, the correlation between PCDH7 expression and Treg signature score (G) and immunosuppressive score (H). Spearman’s correlation test or log-rank test was used for analysis. *p < 0.05
PCDH7 protein negatively correlated with CD8⁺ T-cell infiltration in human PDAC patients. (A) Representative hematoxylin and eosin staining images of human PDAC tissues in the upper row and the corresponding immunofluorescence images of PCDH7 (yellow), AE1/AE3 (sky blue), CD8 (red), and DAPI (blue) in the bottom row. Left: high expression of PCDH7 and low number of CD8 + T-cell image. Right: low expression of PCDH7 and high number of CD8 + T-cell image. Scale bars, 100 μm. (B) Representative images of IHC staining for PCDH7. The intensity score: 0, negative; 1, weak; 2, moderate; and 3, strong. Scale bar, 20 μm. (C) Quantification of the CD8α-positive cell number in PCDH7 low and high cancer cells. (D) Disease-free survival and overall survival analysis (performed using Kaplan–Meier analysis) of PCDH7 (high = 41, low = 28) in human PDAC samples. *p < 0.05
Identification of cold tumor induction–related markers in pancreatic cancer and the clinical implication of PCDH7
  • Article
  • Full-text available

January 2025

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10 Reads

Journal of Cancer Research and Clinical Oncology

Yuki Mochida

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Kenoki Ohuchida

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Bo Zhang

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[...]

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Masafumi Nakamura

Purpose Pancreatic ductal adenocarcinoma (PDAC) is considered a “cold” tumor because the tumor immune microenvironment (TIME) exhibits poor intratumoral T-cell infiltration. This study aimed to identify the marker genes associated with induction of cold TIME in PDAC cells. Methods We orthotopically transplanted 10 primary cultures of PDAC derived from KrasG12D/+; Trp53R172H/+; Pdx-1-Cre (KPC) mice into immunocompetent mice and evaluated TIME by immunohistochemistry (IHC) staining of CD8. We divided primary cultures into two groups: cold TIME group with low CD8⁺ T-cell infiltration and a hot TIME group with high infiltration. RNA sequencing was performed to identify specific genes in the cold TIME group, and single-cell RNA sequencing (scRNA-seq) data was used for validation. IHC was performed to evaluate expressions in human PDAC samples. Results We identified six genes specific in PDAC cells to the cold TIME group by RNA sequencing; these were defined as “cold tumor induction–related genes”. Human PDAC scRNA-seq data revealed that cold tumor induction–related genes were significantly and negatively correlated with the number of CD8⁺ T-cells (p = 0.0341). These genes included protocadherin 7 (PCDH7). High expression of PCDH7 significantly and negatively correlated with the number of CD8⁺ T-cells in scRNA-seq (p = 0.0474) and IHC (p = 0.0110) data using human PDAC samples. PCDH7 was an independent factor for poor prognosis in PDAC (overall survival: hazard ratio = 2.07, p = 0.0367). Conclusion PCDH7 is a prognostic marker associated with CD8⁺ T-cell infiltration for PDAC patients.

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Prognostic impact of subcutaneous fat quality and sarcopenia on the survival outcomes in patients with colorectal cancer

January 2025

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5 Reads

Surgery Today

This study aimed to evaluate the relationship between the quantity and quality of subcutaneous fat and prognosis following colorectal cancer resection. We conducted a retrospective analysis of the clinical data of 399 patients who underwent curative resection for stage 2 or 3 colorectal cancer between January 2013 and March 2019. This study examined the correlation between sarcopenia and various fat parameters, including fat area and density, and assessed their impact on the prognosis. Sarcopenia was associated with a lower subcutaneous and visceral fat area, higher Hounsfield unit value in subcutaneous fat, and reduced modified intramuscular adipose tissue content in the multifidus, erector spinae, and psoas muscles. A low modified intramuscular adipose tissue content in the multifidus and erector spinae muscles was an independent prognostic factor for overall survival (hazard ratio, 2.28; p = 0.0329) and recurrence-free survival (hazard ratio: 2.32, p = 0.0233). Additionally, subcutaneous fat with a high Hounsfield unit was an independent predictor of a recurrence-free survival (hazard ratio, 2.68; p = 0.0142). Subcutaneous fat quality is correlated with sarcopenia and it thus serves as a prognostic factor for recurrence after stage 2 or 3 colorectal cancer resection.


Tumor infiltration of inactive CD8 + T cells was associated with poor prognosis in Gastric Cancer

December 2024

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10 Reads

Gastric Cancer

Background Gastric cancer (GC) shows limited response to immune checkpoint inhibitors due to its complex tumor immune microenvironment (TIME). This study explores the functions of various immune cells in the complex TIME in GC. Methods We assessed CD8 + T-cell infiltration of GC tissues by immunohistochemistry, and performed single-cell RNA sequencing (scRNA-seq) of tumor and normal tissues from 34 patients with GC. Results We categorized 157 GC patients into LOW, MID, and HIGH groups based on their CD8 + T-cell infiltration. Overall survival was notably lower for the HIGH and LOW groups compared with the MID group. Our scRNA-seq data analysis showed that CD8 + T-cell activity markers in the HIGH group were expressed at lower levels than in normal tissue, but the T-cell-attracting chemokine CCL5 was expressed at a higher level. Notably, CD8 + T-cells in the HIGH group displayed lower PD1 expression and higher CTLA4 expression. TCR repertoire analysis using only Epstein–Barr virus-negative cases showed that CD8 + T-cell receptor clonality was lower in the HIGH group than in the MID group. Furthermore, in the HIGH group, the antigen-presenting capacity of type 1 conventional dendritic cells was lower, the immunosuppressive capacity of myeloid-derived suppressor cells was higher, and the expression of CTLA4 in regulatory T-cells was higher. Conclusion The present data suggest that the infiltration of inactive CD8 + T-cells with low clonality is induced by chemotaxis in the HIGH group, possibly leading to a poor prognosis for patients with GC.


Single‐cell atlas of CRC patients with/without renal transplantation. (A) Schematic diagram of scRNA‐seq analysis design and workflow. UMAP of 87,116 single cells after quality check, normalization, and exclusion of doublets colors by clusters (B) and grouped by spatial distribution of cells in non‐transplant and renal transplantation tissues (C). (D) Feature plots highlighting the expression of canonical markers for identified cell types. (E) UMAP for cell type delineation based on marker gene expression. (F) Heatmap showing the representative predominant genes characterizing each cell type. (G) Bar plot illustrating the relative abundance of each cell type within the study groups. CRC, colorectal cancer; scRNA‐seq, single‐cell RNA sequencing; UMAP, uniform manifold approximation and projection.
Visualization of the specific immune cells in samples from the non‐transplant and renal transplantation groups. (A) mIF depicting the distribution of CD3⁺, CD20⁺, CD11c⁺, and CD68⁺ cells. (B) Representative IHC images and statistical analysis (C) of CD8⁺, GZMB⁺, and FOXP3⁺ cells within tumor lesions. (D) Representative IHC images and statistical analysis (E) of CD8⁺, GZMB⁺, and FOXP3⁺ cells within normal areas (ns not significant). IHC, immunohistochemistry; mIF, multiplex immunofluorescence; ns, not significant.
CD8⁺ T cell clustering and transcriptomic alterations. (A) UMAP visualization of CD8⁺ T cells grouped by subtypes. (B) UMAP visualization of CD8⁺ T cells grouped by spatial distribution in non‐transplant and renal transplantation tissues. (C) Dot plot displaying marker genes across CD8⁺ T cell clusters. Dot size indicates the fraction of expressing cells, colored by Z score‐normalized expression levels. (D) Heatmap of scaled normalized expression for functional pathway genes in CD8⁺ T cell clusters. (E) Bar plot of cell cluster frequency representation among groups. (F) Comparison of signature gene expression in tumor tissue between the non‐transplant and renal transplantation groups. The violin plots were drawn based on each cell's scored signature gene expression. (G) and (H) Comparison of functional gene expression scores in CD8⁺GZMH and CD8⁺GZMB subtypes. The violin plots were drawn based on the scored signature gene (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). nRT, non‐renal transplantation; ns, not significant; RT, renal transplantation; UMAP, uniform manifold approximation and projection.
CD4⁺ T cell clustering and transcriptomic alterations. (A) UMAP visualization of CD4⁺ T cells grouped by subtypes. (B) UMAP visualization of CD4⁺ T cell grouped by spatial distribution in non‐transplant and renal transplantation tissues. (C) Dot plot displaying marker genes across CD4⁺ T cell clusters. Dot size indicates the fraction of expressing cells, colored by Z score‐normalized expression levels. (D) Bar plot of cell cluster frequency representation among groups. (E) Comparison of signature gene expression in tumor tissue between non‐transplant and renal transplantation groups. The violin plots were drawn based on each cell's scored signature gene expression. (F) Comparison of functional gene expression scores in CD4⁺FOXP3 subtype. The violin plots were drawn based on the scored signature gene. (G) Representative IHC images and the statistical result of CTLA4⁺ cell abundance in the non‐transplant and renal transplantation group tumor lesions. (H) Representative mIHC images of CD8⁺CTLA4⁺ cells and FOXP3⁺CTLA4⁺ cells (Tregs) using antibodies against CTLA4 (red), CD8 (green), and FOXP3 (light blue) in renal transplantation tumors; nuclei are indicated in blue. The white arrow indicates CD8⁺CTLA4⁺ cells (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). IHC, immunohistochemistry; mIHC, multiplex immunohistochemistry; nRT, non‐renal transplantation; ns, not significant; RT, renal transplantation; UMAP, uniform manifold approximation and projection.
B Cell clustering and transcriptomic alterations. (A) UMAP visualization of B cells grouped by subtypes. (B) UMAP visualization of B cells grouped by spatial distribution in non‐transplant and renal transplantation tissues. (C) Dot plot displaying marker genes across B cell clusters. Dot size indicates the fraction of expressing cells, colored by Z score‐normalized expression levels. (D) Heatmap of scaled normalized expression for functional pathway genes in B cell clusters. (E) Bar plot of cell cluster frequency representation among groups. (F) Comparison of signature gene expression in tumor tissue between non‐transplant and renal transplantation groups. The violin plots were drawn based on each cell's scored signature gene expression. (G) Comparison of functional gene expression scores in the activated B cell subtype. The violin plots were drawn based on the scored signature gene (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). nRT, non‐renal transplantation; ns, not significant; RT, renal transplantation; UMAP, uniform manifold approximation and projection.
Exploring the tumor microenvironment of colorectal cancer patients post renal transplantation by single‐cell analysis

December 2024

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19 Reads

Patients with colorectal cancer (CRC) following renal transplantation require long‐term immunosuppressants to prevent graft rejection. However, the impact of these immunosuppressants on the tumor immune microenvironment and the roles of immune cells within it remain poorly understood. We conducted comprehensive single‐cell RNA sequencing on tumor and normal tissues from four CRC patients post renal transplantation and compared these with published data from 23 non‐transplant CRC patients. We set four groups for detailed comparative analysis based on the renal transplantation status and tissue origin: non‐renal transplantation normal (nRT_Normal), non‐renal transplantation tumor (nRT_Tumor), renal transplantation normal (RT_Normal), renal transplantation tumor (RT_Tumor). Our analysis revealed significant tumor immune microenvironment landscape alterations in the transplantation group. CD8⁺effector T cells of RT_Tumor showed significantly diminished cytotoxicity and tumor neoantigen recognition (p < 0.0001), while CD4⁺FOXP3 regulatory T cells of RT_Tumor displayed a higher inhibitory score (p < 0.05), indicating preserved immunomodulatory potential compared with non‐transplant CRC. Notably, significantly increased CTLA4 expression in T cells of RT_Tumor was found and testified (p < 0.05). Our findings provide novel mechanistic insights for understanding the immune landscape in renal transplant recipients with CRC and pave the way for potential immunotherapeutic strategies that may improve survival and quality of life for this patient population.


TIM3 on natural killer cells regulates antibody-dependent cellular cytotoxicity in HER2-positive gastric cancer

December 2024

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71 Reads

Cancer Letters

Therapies targeting HER2 are the standard treatment for HER2-positive gastric cancer (GC). Trastuzumab, a monoclonal antibody against HER2, exerts anti-tumor activity through cell growth regulation and antibody-dependent cellular cytotoxicity (ADCC). ADCC is induced by the binding of trastuzumab to Fcγ receptor III (CD16) in natural killer (NK) cells. However, the relationship between immune checkpoint (IC) molecules of NK cells and trastuzumab-induced ADCC is poorly understood. We performed single-cell RNA sequencing (scRNA-seq) and immunohistochemistry to identify IC molecules associated with CD16 expression in NK cells of GC patients. Additionally, we conducted in vitro assays with HER2-transfected GC cells and in vivo experiments using a mouse HER2-positive GC model to assess expression changes in IC molecules in NK cells and their ligands during trastuzumab treatment. In GC patients, the expression of TIM3, an IC molecule, was strongly correlated with that of CD16 in NK cells. In vitro assays showed that ADCC with trastuzumab increased TIM3 expression in NK cells. scRNA-seq analysis revealed that TIM3 expression of cytotoxic NK cells was elevated in HER2-positive GC patients treated with trastuzumab. HMGB1, a TIM3 ligand, was expressed at higher levels in HER2-transfected GC cells than in controls. Furthermore, HMGB1 expression was higher in HER2-positive GC patients treated with trastuzumab compared to untreated HER2-positive GC patients. In the mouse HER2-positive GC model, anti-TIM3 antibodies and trastuzumab demonstrated synergistic anti-tumor effects without toxicity. This study suggests the combined anti-TIM3 antibody and trastuzumab therapy may have potential as a new treatment strategy for HER2-positive GC.


Dynamic glycolytic reprogramming effects on dendritic cells in pancreatic ductal adenocarcinoma

September 2024

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32 Reads

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1 Citation

Journal of Experimental & Clinical Cancer Research

Background: Pancreatic ductal adenocarcinoma tumors exhibit resistance to chemotherapy, targeted therapies, and even immunotherapy. Dendritic cells use glucose to support their effector functions and play a key role in anti-tumor immunity by promoting cytotoxic CD8+ T cell activity. However, the effects of glucose and lactate levels on dendritic cells in pancreatic ductal adenocarcinoma are unclear. In this study, we aimed to clarify how glucose and lactate can impact the dendritic cell antigen-presenting function and elucidate the relevant mechanisms. Methods: Glycolytic activity and immune cell infiltration in pancreatic ductal adenocarcinoma were evaluated using patient-derived organoids and resected specimens. Cell lines with increased or decreased glycolysis were established from KPC mice. Flow cytometry and single-cell RNA sequencing were used to evaluate the impacts on the tumor microenvironment. The effects of glucose and lactate on the bone marrow-derived dendritic cell antigen-presenting function were detected by flow cytometry. Results: The pancreatic ductal adenocarcinoma tumor microenvironment exhibited low glucose and high lactate concentrations from varying levels of glycolytic activity in cancer cells. In mouse transplantation models, tumors with increased glycolysis showed enhanced myeloid-derived suppressor cell infiltration and reduced dendritic cell and CD8+ T cell infiltration, whereas tumors with decreased glycolysis displayed the opposite trends. In three-dimensional co-culture, increased glycolysis in cancer cells suppressed the antigen-presenting function of bone marrow-derived dendritic cells. In addition, low-glucose and high-lactate media inhibited the antigen-presenting and mitochondrial functions of bone marrow-derived dendritic cells. Conclusions: Our study demonstrates the impact of dynamic glycolytic reprogramming on the composition of immune cells in the tumor microenvironment of pancreatic ductal adenocarcinoma, especially on the antigen-presenting function of dendritic cells.


Patients’ flow chart. The medical records of 1,471 consecutive patients preoperatively diagnosed with colorectal cancer (CRC) and underwent resection were reviewed. Finally, 1,272 patients met the inclusion criteria. Multiple CRCs were categorized into synchronous and metachronous cohorts. CRC colorectal cancer, MIS minimally invasive surgery, FAP familial adenomatous polyposis
Schema of the patterns of metachronous colorectal cancers after resection of left-sided colon or rectal cancers. A-1 the first operation of pattern A is for left-sided colon or rectal cancer without left colic artery (LCA) preservation. The second operation is for right-sided colon cancer. A-2 the second operation is for left-sided colon or rectal cancer, requiring the resection of previous anastomotic site. B-1 the first operation of pattern B is for left-sided colon or rectal cancer with LCA preservation. The second operation is for right-sided colon cancer. B-2 and B-3 the second operation is for left-side colon or rectal cancer. Pattern B-2 does not require the resection of the previous anastomotic site owing to the preserved LCA. ICA ileocolic artery, rtMCA right branch of middle colic artery, ltMCA left branch of middle colic artery, IMA inferior mesenteric artery, LCA left colic artery
Schema of the patterns of metachronous colorectal cancers after resection of right-side colon cancers. C Pattern C is left-side colon or rectal cancer after right-side colon resection. D Pattern D is right-side colon cancer after transverse colectomy, requiring the previous anastomotic site resection. ICA ileocolic artery, rtMCA right branch of middle colic artery, IMA inferior mesenteric artery
Clinical features, surgical treatment strategy, and feasibility of minimally invasive surgery for synchronous and metachronous multiple colorectal cancers: A 14-year single-center experience

September 2024

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40 Reads

Surgical Endoscopy

Background Patients with a history of colorectal cancer (CRC) are at increased risk of developing secondary synchronous/metachronous CRCs. The role of minimally invasive surgery (MIS) for multiple CRCs remains unclear. This study aimed to evaluate the short-term outcomes of MIS in patients with multiple CRCs and elucidate their clinical characteristics. Methods This retrospective study reviewed CRC patients who underwent MIS between 2010 and 2023. Multiple CRC cases were categorized into synchronous and metachronous cohorts. Demographics, pathological findings, and perioperative outcomes were analyzed. Propensity score matching (PSM) analysis was performed as appropriate. Results A total of 1,272 patients met the inclusion criteria, with 99 (7.8%) having multiple CRCs (75 synchronous and 24 metachronous). Multiple CRC patients had a higher prevalence of strong family history (8.1% vs. 1.0%, P < 0.001) and right-sided colon cancer (55.6% vs. 34.4%, P < 0.001) compared to solitary CRC patients. MSI-high/MMR-deficient status, including Lynch syndrome, was frequently observed among patients with multiple CRCs. Synchronous CRCs requiring double-anastomosis were associated with longer operation times (P = 0.03) and increased blood loss (P = 0.03) compared to those with a single-anastomosis. In the metachronous cohort, repeat operation patterns were categorized based on tumor location and sacrificed arteries. Preservation of the left-colic artery avoided extended colectomy in some patients. Patients with multiple CRC involving rectal cancer had a higher anastomotic leakage (AL) rate (17.6% vs. 5.7%, P < 0.01); however, this difference in AL rate disappeared after PSM (8.8% vs. 8.8%, P = 1.0). In patients with multiple CRCs, AL has not been observed ever since the indocyanine green fluorescence imaging was implemented. Conclusions MIS is feasible for multiple CRCs, with perioperative outcomes comparable to those for solitary CRCs. Preservation of critical arteries may benefit patients at high risk of secondary CRCs, particularly those with a strong family history of CRC, right-sided tumors, or MSI-high/MMR-deficient profiles, including Lynch syndrome. Graphical abstract


Combined Autophagy Inhibition and Dendritic Cell Recruitment Induces Antitumor Immunity and Enhances Immune Checkpoint Blockade Sensitivity in Pancreatic Cancer

September 2024

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65 Reads

Cancer Research

The effect of immune checkpoint inhibitors is extremely limited in patients with pancreatic ductal adenocarcinoma (PDAC) due to the suppressive tumor immune microenvironment. Autophagy, which has been shown to play a role in antitumor immunity, has been proposed as a therapeutic target for PDAC. In this study, single-cell RNA sequencing of autophagy-deficient murine PDAC tumors revealed that autophagy inhibition in cancer cells induced dendritic cell (DC) activation. Analysis of human PDAC tumors substantiated a negative correlation between autophagy and DC activation signatures. Mechanistically, autophagy inhibition increased the intracellular accumulation of tumor antigens, which could activate DCs. Administration of chloroquine, an autophagy inhibitor, in combination with Flt3 ligand–induced DC infiltration inhibited tumor growth and increased tumor-infiltrating T lymphocytes. However, autophagy inhibition in cancer cells also induced CD8+ T-cell exhaustion with high expression of immune checkpoint LAG3. A triple-therapy comprising chloroquine, Flt3 ligand, and an anti-LAG3 antibody markedly reduced tumor growth in orthotopic syngeneic PDAC mouse models. Thus, targeting autophagy in cancer cells and activating DCs sensitize PDAC tumors to immune checkpoint inhibitor therapy, warranting further development of this treatment approach to overcome immunosuppression in pancreatic cancer. Significance: Inhibiting autophagy in pancreatic cancer cells enhances intracellular accumulation of tumor antigens to induce dendritic cell activation and synergizes with immunotherapy to markedly inhibit the growth of pancreatic ductal adenocarcinoma.


Figure 1. TAK1 activation is higher in CAFs in PDAC, associating with immunosuppressive markers, and TAK1 + CAFs are far from the α-SMA + phenotype CAFs. A, Box plots showing expression levels of TAK1 in PDAC tumors compared with normal samples in TCGA and GTEx data; n ¼ 179 patients (tumor); n ¼ 171 (normal). This analysis was conducted using one-way ANOVA. B, Representative co-IF image of TAK1 (red) and CK19 (green) in PDAC. C, Representative IHC staining of TAK1 in human PDAC tissues. Scale bar, 100 µm. D, Representative images of TAK1 (green) and PDPN (red) co-IF staining (left) and TAK1 (green) and FAP (red) co-IF staining (right) in PDAC. Scale bar, 100 µm. E, Western blots showing TAK1 expression in PDAC cell lines and CAFs. F, Correlation scatter plot with the Pearson coefficient (R) of the T-cell exhaustion signature (LAG3, PDCD1, CTLA4, HAVCR2, and TIGHT) vs MAP3K7 (log 2 RNA-seqV2, BE norm.) in the PDAC samples from TCGA database. G, Inverse correlation between TAK1 and α-SMA expression in CAF-infiltrating tumors in human PDAC slices. TAK1 (red) and α-SMA (green) expression in tumors in human PDAC slices were examined with IF staining; scale bar, 100 µm. Boxed areas (a-c) are magnified in adjacent panels, showing different levels of TAK1 and α-SMA expression in CAFs from patient sections. H, The CAFs were categorized into α-SMA + and α-SMA � groups, and the number of TAK1-positive cells was detected and quantified (as shown in G). TAK1 positivity was analyzed and quantified in 50 randomly selected high-power fields (400�) from five patients with PDAC (P < 0.01; unpaired Student t test ). I, Representative co-IF image of TAK1 (green), a-SMA (green), and CK19 (red). The images on the right are enlarged views. The white lines represent the tumor edge and CAF edge. Arrows indicate the vertical distance between the two white lines. TAK1 + cells infiltrated into the stroma, where α-SMA + CAFs were near the cancer gland. Counterstaining with DAPI (blue). Bar, 100 μm (bottom). J, Quantification calculation of the vertical distance between TAK1 + CAFs with CK19 and between α-SMA + CAFs with CK19 in the stroma. Results show mean ± SD of five human tissues. ****, P < 0.0001, unpaired Student t test. DAPI, 4 0 ,6-diamidino-2-phenylindole; FAP, fibroblast activation protein; GTEx, Genotype-Tissue Expression; PAAD, pancreatic adenocarcinoma; PDPN, podoplanin.
Figure 4. Interference of TAK1 in CAFs decreases CAF-led cancer cell invasion and restricts PDO outgrowth. A, Schematic representation of 3D direct coculture models. GFP-tagged PDOs were seeded at a 1:1 ratio (1.25 � 10 4 PDO cells and 1.25 � 10 4 CAFs/well) into 96-well plates with Matrigel and incubated with a control medium. B, Bright field of representative images of PDOs. Bar, 100 μm (bottom). C, Spheroids containing PDO497-GFP (green) and NTCAF-RFP (red) or si#1TAK1 CAFs-RFP (red) or si#2TAK1 CAFs-RFP (red) embedded in the Matrigel matrix for 3 days. Scale bars, 100 μm. D, Quantification of the total area of fibroblast spheroids and PDO growth per spheroid (*, P < 0.05; **, P < 0.01; ****, P < 0.0001). E, PDOs were directly cocultured with CAFs that were transfected with negative control siRNA or siTAK1 RNAs. Areas of fibroblast-led invasion (green) or cancer cell outgrowth (yellow) per spheroid are shown in the images. Scale bars, 100 μm. F, Overlays illustrate the definition of the area of outgrowth of the spheroid (yellow) and the area of fibroblast-led invasion (green) according to the morphologic appearance of cells under bright-field microscopy. Quantification of the total spheroid area per main body area (green in E) or outgrowth area (yellow in E) was normalized to the ratio to the mean value of each spheroid in the nontreated group (n ¼ 5; data were analyzed using an unpaired Student t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001). (A, Created with BioRender.com.)
Figure 7. Model explaining the role of TAK1 in CAFs in the PDAC microenvironment. TAK1 + CAFs secrete inflammatory factors such as IL6 and CXCL1 to stimulate tumor growth. TAK1 + CAFs are distanced from the tumor and associated with immunosuppression (left). Interference with TAK1 in CAFs leads to decreased proliferation, reduced migration and invasion, downregulation of oncogene and inflammatory factors through the MAPK/NF-κB pathway, and limited tumor EMT. CAFs lacking TAK1 have higher stiffness and are closer to the tumor, restricting its growth. Additionally, increased CD4 and CD8 T-cell infiltration is observed, whereas suppressive immune cells decrease, improving the PDAC immune microenvironment (right). ECM, extracellular matrix. (Created with BioRender.com.)
TAK1 Promotes an Immunosuppressive Tumor Microenvironment through Cancer-Associated Fibroblast Phenotypic Conversion in Pancreatic Ductal Adenocarcinoma

September 2024

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26 Reads

Clinical Cancer Research

Purpose We aim to clarify the precise function of TGFβ1-activated kinase 1 (TAK1) in cancer-associated fibroblasts (CAF) within human pancreatic ductal adenocarcinoma (PDAC) by investigating its role in cytokine-mediated signaling pathways. Experimental Design The expression of TAK1 in pancreatic cancer was confirmed by The Cancer Genome Atlas data and human pancreatic cancer specimens. CAFs from freshly resected PDAC specimens were cultured and used in a three-dimensional model for direct and indirect coculture with PDAC tumors to investigate TAK1 function. Additionally, organoids from [LSL-KrasG12D/+, LSL-Trp53R172H/+, Pdx1-Cre (KPC)] mice were mixed with CAFs and injected subcutaneously into C57BL/6 mice to explore in vivo functional interactions of TAK1. Results The Cancer Genome Atlas data revealed significant upregulation of TAK1 in PDAC, associating with a positive correlation with the T-cell exhaustion signature. Knockdown of TAK1 in CAFs decreased the inflammatory CAF signature and increased the myofibroblastic CAF signature both in vitro and in vivo. The absence of TAK1 hindered CAF proliferation, blocked several inflammatory factors via multiple pathways associated with immunosuppression, and hindered epithelial–mesenchymal transition and outgrowth in vitro in spheroid cocultures with PDAC cells. Additionally, TAK1 inhibitor restrained tumor growth, increased CD4+ and CD8+ T-cell abundance, and reduced immunosuppressive cells present in vivo. Conclusions Blocking the TAK1+ CAF phenotype leads to the conversion of protumorigenic CAFs to antitumorigenic CAFs. This highlights TAK1 as a potential therapeutic target, particularly in CAFs, and represents a novel avenue for combined immunotherapy in PDAC.


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Tumor-associated neutrophils upregulate Nectin2 expression, creating the immunosuppressive microenvironment in pancreatic ductal adenocarcinoma

September 2024

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32 Reads

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2 Citations

Journal of Experimental & Clinical Cancer Research

Background Tumor-associated neutrophils (TANs) constitute an abundant component among tumor-infiltrating immune cells and have recently emerged as a critical player in pancreatic ductal adenocarcinoma (PDAC) progression. This study aimed to elucidate the pro-tumor mechanisms of TAN and identify a novel target for effective immunotherapy against PDAC. Methods Microarray and cytokine array analyses were performed to identify the mechanisms underlying the function of TANs. Human and mouse TANs were obtained from differentiated HL-60 cells and orthotopically transplanted PDAC tumors, respectively. The interactions of TANs with cancer and cytotoxic T-cells were evaluated through in vitro co-culture and in vivo orthotopic or subcutaneous models. Single-cell transcriptomes from patients with PDAC were analyzed to validate the cellular findings. Results Increased neutrophil infiltration in the tumor microenvironment was associated with poor survival in patients with PDAC. TANs secreted abundant amounts of chemokine ligand 5 (CCL5), subsequently enhancing cancer cell migration and invasion. TANs subpopulations negatively correlated with cytotoxic CD8 ⁺ T-cell infiltration in PDAC and promoted T-cell dysfunction. TANs upregulated the membranous expression of Nectin2, which contributed to CD8 ⁺ T-cell exhaustion. Blocking Nectin2 improved CD8 ⁺ T-cell function and suppressed tumor progression in the mouse model. Single-cell analysis of human PDAC revealed two immunosuppressive TANs phenotypes: Nectin2 ⁺ TANs and OLR1 ⁺ TANs. Endoplasmic reticulum stress regulated the protumor activities in TANs. Conclusions TANs enhance PDAC progression by secreting CCL5 and upregulating Nectin2. Targeting the immune checkpoint Nectin2 could represent a novel strategy to enhance immunotherapy efficacy in PDAC.


Citations (50)


... The immunosuppressive TME of PAAD promotes immune evasion by tumor cells, involving various cell types, cytokines, and other components that impair anti-tumor immunity, representing a key obstacle to the advancement of pancreatic cancer treatment [29][30][31]. Immunosuppressive cells, including M2 macrophages, Tregs, and myeloid-derived suppressor cells (MDSCs), play a critical role in maintaining the immunosuppressive TME [32][33][34]. In our current study, compared to samples with low NIPAL1 expression, those with high expression exhibited a higher proportion of Macrophages M0, Macrophages M1, Dendritic cells resting, Tregs, Neutrophils, T cells CD4 memory resting, T cells CD4 memory activated and Eosinophils. ...

Reference:

NIPAL1 as a prognostic biomarker associated with pancreatic adenocarcinoma progression and immune infiltration
Dynamic glycolytic reprogramming effects on dendritic cells in pancreatic ductal adenocarcinoma

Journal of Experimental & Clinical Cancer Research

... Histamine receptors are highly expressed in various malignant tumor tissues, such as those of the breast, bowel, pancreas, and prostate. They are positively correlated with the clinical stage of the tumor and negatively correlated with patient prognosis (23,24). ...

Blockade of histamine receptor H1 augments immune checkpoint therapy by enhancing MHC-I expression in pancreatic cancer cells

Journal of Experimental & Clinical Cancer Research

... Takashi Taniguchi 2024 Japan [20] via the 'midas' command. Utilizing the "midas" feature of the random-effects model, estimations were made for combined sensitivity, specificity, SROC curves, combined positive and negative likelihood ratios, and diagnostic advantage ratios, varying with biomarker types (like platelet-derived EVs, cell surface proteoglycan, Exocet), surface proteoglycan, exosomal circular RNA, exomiR, exosome source, exosome isolation method, and biomarker analysis approach. ...

MicroRNA‐20a in extracellular vesicles derived from duodenal fluid is a possible biomarker for pancreatic ductal adenocarcinoma

... Accordingly, subcutaneous emphysema (SE) as a postoperative complication has garnered attention because a higher incidence of SE in robotic surgery than in laparoscopic surgery has been suggested (33.7-41.2% vs. 17.5-19.0%) [2,3]. This sometimes develops into severe SE, which can lead to extubation failure or respiratory disorders [2,4,5]. ...

Risk factors and clinical significance of subcutaneous emphysema after robot-assisted laparoscopic rectal surgery: a single-center experience

Journal of Robotic Surgery

... MDSCs suppress T-cell activity and create an immunosuppressive environment, contributing to resistance to immunotherapy in GC [60] (Fig. 2C). Studies have shown that in the GC TME, infiltrating monocytic MDSCs (M-MDSCs) highly express immunosuppressive genes and are significantly enriched in GC tissues, with IER3 + M-MDSCs being closely associated with immunosuppression and treatment resistance [61]. ...

Tumor-infiltrating monocytic myeloid-derived suppressor cells contribute to the development of an immunosuppressive tumor microenvironment in gastric cancer
  • Citing Article
  • January 2024

Gastric Cancer

... Previously, it was considered challenging to transmit surgical skills remotely [10]. However, in the field of robotic surgery, successful attempts at remote mentoring, particularly through tele-mentoring, have demonstrated the viability and effectiveness of remote instruction [11,12]. Additionally, the physical constraints imposed by the COVID-19 lockdown accelerated the adoption of remote education, showing that web-based learning can be as effective as in-person learning [13][14][15][16]. ...

Real-time telementoring with 3D drawing annotation in robotic surgery

Surgical Endoscopy

... Furthermore, CXCL13 was most highly expressed in the EBV-negative HIGH group ( Fig. 3I (Fig. S3I), whereas CXCR5 expression in B-cells was highest in the MID group (Fig. S3J). The CXCL13-CXCR5 axis is involved in ectopic or tertiary lymphoid structure (TLS) formation [18]. ...

Tertiary lymphoid structures correlate with enhancement of antitumor immunity in esophageal squamous cell carcinoma
  • Citing Article
  • August 2023

British Journal of Cancer

... When considering the efficacy of biologics for CD treatment, a low SMI may also impair immune function and wound healing of CD patients, leading to an increased risk of complications and surgery [26]. Ando K. et al. were the first to investigate the association between logics in CD patients-findings with potential implications for the pathophysiology and treatment of CD. ...

Strong impact of sarcopenic state defined by skeletal muscle mass index on postoperative complication of Crohn's disease patients

Surgery Open Science

... Of note, increasing evidence points to bacteria as possible contributors to PDAC development (11,12). Recent clinical and experimental investigations have demonstrated the presence of an intratumor microbiome in PDAC and revealed oncological actions of some species of bacteria (11)(12)(13)(14)(15)(16)(17). Accumulated ...

Intratumor Fusobacterium nucleatum promotes the progression of pancreatic cancer via the CXCL1-CXCR2 axis

... The clinical significances of the components of the TME, such as tumor-infiltrating lymphocytes, intratumoral CD8 + cells, PD-L1 expression status, and tumorassociated macrophages, have been studied in ESCC patients. [12][13][14] The TME status and its associations with clinical outcomes have been evaluated mainly in ESCC patients not receiving NAC. 15 Recent studies have examined the effects of NAC on TME in ESCC, [16][17][18] and highlighted the impacts of tumor-infiltrating lymphocytes and PD-L1 expression on the clinical outcomes of ESCC patients receiving NAC followed by surgery. 18,19 The clinical impacts of various TME ...

Neoadjuvant chemotherapy enhances anti-tumor immune response of tumor microenvironment in human esophageal squamous cell carcinoma
  • Citing Article
  • March 2023

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