Journal: International Journal of Surgery (London, England)

Article Title: Prediction values of tertiary lymphoid structures in the prognosis of patients with left- and right-sided colon cancer: a multicenter propensity score-matched study

doi: 10.1097/JS9.0000000000000483

Figure Lengend Snippet: H&E sections showing TLS and different immune cells in TLS. (A) Representative figure of H&E slides to show TLS. (B-K) IHC results showed the different immune cells, which formed the TLS, including (B) CD4 + T cells, (C) CD8 + T cells, (D) CD20 + B cells, (E) CD45RO + memory T cells, (F) CD21 + follicular dendritic cells, (G) CD11c + dendritic cells, (H) CD15 + granulocytes, (I) CD68 + macrophages, (J) FOXP3 + Treg cells, and (K) NCR1 + natural killer cells. (L and M) Pie chart figure showed the distribution of various immune cells forming the TLS between LCC and RCC. (N–P) Scatter plots showed different TLS maturity status between LCC and RCC. LCC, left-sided colon cancer; RCC, right-sided colon cancer.

Article Snippet: IHC staining was performed to identify helper T cells (Abcam, ab133616, rabbit monoclonal to CD4, 1:200), cytotoxic T cells (Abcam, ab178089, rabbit monoclonal to CD8 alpha, 1:100), regulatory T cells (Abcam, ab20034, mouse monoclonal to FOXP3, 1:500), memory T cells (Abcam, mouse monoclonal to CD45RO, 1:1,000), B cells (Abcam, ab78237, rabbit monoclonal to CD20, 1:100), dendritic cells (Abcam, ab52632, rabbit monoclonal to CD11c, 1:500), natural killer cells (Abcam, ab224703, rabbit monoclonal to NCR1, 1:1000), follicular dendritic cells (Abcam, ab75985, rabbit monoclonal to CD21, 1:100), macrophage (Wuhan Sevicebio Technology Co., Ltd, GB113150, rabbit monoclonal to CD68, 1:500), and tumor-associated neutrophil (Santa Cruz, SC-21702, mouse monoclonal to CD15, 1:100).

Techniques:

Journal: Nature Communications

Article Title: UPP1 promotes lung adenocarcinoma progression through the induction of an immunosuppressive microenvironment

doi: 10.1038/s41467-024-45340-w

Figure Lengend Snippet: a mIF of PanCK/UPP1 (UPP1 high/low tumor cells), and CD4/FOXP3 (FOXP3 + CD4 + T cells) ( n = 15), representative images shown. b mIF of PanCK/UPP1 (UPP1 high/low tumor cells), and α-SMA/MMP11 (MMP11+ fibroblasts) ( n = 15), representative images shown. c mIF of PanCK/UPP1 (UPP1 high/low tumor cells), and CD8/LAG3/PD-1 (LAG3 + PD-1 + CD8 + T cells) ( n = 15), representative images shown. d mIF of PanCK/UPP1 (UPP1 high/low tumor cells), and CD68/SPP1 (SPP1 + CD68+ macrophages) ( n = 15), representative images shown. The image on the far left represents a panoramic scan, with the ROIs indicating the selected areas. Scale bars = 5 mm. The image on the upper right represents a magnified portion of the white square in the panoramic scan on the left, which is displayed as an original image, classified image, and merged image. Scale bars = 200 μm. The images below represent further magnified views of each antibody staining channel from the selected area. Scale bars = 25 μm. For the ROIs of each section, the spatial distances between the tumor cells and the immune/fibroblast cells were calculated.

Article Snippet: The purity of CD4 + T cells was validated using flow cytometry; Untouched CD8 + T cells were purchased from iXCells Biotechnologies (10HU-024N); CD4 + T cells or CD8 + T cells were cultured in ImmunoCult T Cell Expansion Medium (STEMCELL, 10981) supplemented with 50 U/ml IL-2 (Peprotech, 200-02-50) and ImmunoCult CD3/CD28/CD2 T cell Activator (STEMCELL, 10970).

Techniques: Staining

Journal: Nature Communications

Article Title: UPP1 promotes lung adenocarcinoma progression through the induction of an immunosuppressive microenvironment

doi: 10.1038/s41467-024-45340-w

Figure Lengend Snippet: a 80-cytokine array analysis between UPP1-overexpressing HCC827 tumor cells (UPP1-OE) and control cells (UPP1-OENC) ( n = 3). b Top six upregulated cytokines in HCC827 UPP1-OE tumor cells ( n = 3). c ELISA comparison of TGF-β1 expression in culture supernatants of HCC827 UPP1-OE and UPP1-OENC tumor cells ( n = 4). d Co-culture workflow, created with BioRender.com. αTGF-β1, TGF-β1 neutralizing antibodies; IgG, isotype control IgG. e Flow cytometry analysis of CD25 + FOXP3+ Tregs proportion ( n = 4). f Flow cytometry analysis of LAG3 + PDCD1 + CD8 + T cells proportion ( n = 4). g Representative immunofluorescence (IF) staining of SPP1 in THP-1-derived macrophages ( n = 4). MFIs represent mean fluorescence intensities. Scale bars = 20 μm, h Western blot analysis of SPP1 expression in THP-1-derived macrophages after co-culture ( n = 3). i RT-qPCR measurement of M2 macrophage markers in THP-1-derived macrophages post co-culture ( n = 3). j scRNA-seq analysis of fibroblasts after co-culturing with HCC827 UPP1-OE tumor cells/UPP1-OENC tumor cells. UPP1-OE tumor cells = 7707; UPP1-OENC tumor cells = 8200. k Comparison of CAFs scores in fibroblasts co-cultured with either UPP1-OE tumor cells or UPP1-OENC tumor cells. l Dot plot showing the mean expression of CAFs marker genes. m The UMAP plot displaying the distribution of the MMP11+ CAFs scores. Fibroblasts with an MMP11+ CAFs score higher than the median value were categorized as MMP11+ CAFs-like fibroblasts, while the others were defined as MMP11- fibroblasts (Fbs). n Comparison of the proportion of MMP11+ CAFs-like fibroblasts between the UPP1-OE tumor cells group and the UPP1-OENC tumor cells group. o Comparison of the CAFs-related biological processes between the UPP1-OE tumor cells group and the UPP1-OENC tumor cells group. p Western blot analysis of CAFs markers after the treatment of TGF-β1 neutralizing antibodies ( n = 3). n denotes biologically independent samples. Data were represented as mean ± SD in b , c , e – g . Data were represented as mean ± SEM in i . Two-tailed student’s t -test was used in b , c ; one-way ANOVA with multiple comparisons in e – g , i ; two-tailed Wilcoxon rank-sum test in k and o . Two-tailed Fisher’s exact test in n . Source data are provided as a Source Data file.

Article Snippet: The purity of CD4 + T cells was validated using flow cytometry; Untouched CD8 + T cells were purchased from iXCells Biotechnologies (10HU-024N); CD4 + T cells or CD8 + T cells were cultured in ImmunoCult T Cell Expansion Medium (STEMCELL, 10981) supplemented with 50 U/ml IL-2 (Peprotech, 200-02-50) and ImmunoCult CD3/CD28/CD2 T cell Activator (STEMCELL, 10970).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Comparison, Expressing, Co-Culture Assay, Flow Cytometry, Immunofluorescence, Staining, Derivative Assay, Fluorescence, Western Blot, Quantitative RT-PCR, Cell Culture, Marker, Two Tailed Test

Journal: Nature Communications

Article Title: UPP1 promotes lung adenocarcinoma progression through the induction of an immunosuppressive microenvironment

doi: 10.1038/s41467-024-45340-w

Figure Lengend Snippet: a Two-tailed Pearson correlation analysis between UPP1 and immune checkpoints across different datasets, including the scRNA-seq dataset (cells = 49,113), bulk datasets (bCohort1, n = 559; bCohort2, n = 398; bCohort3, n = 572), and CCLE mRNA ( n = 440) and protein expression ( n = 37) datasets. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. b Representative images of IHC staining of UPP1 and PD-L1 in our TMA cohort from Zhongshan Hospital ( n = 205). Scale bars = 100 μm. c Two-tailed Pearson correlation between UPP1 and PD-L1 in our TMA cohort ( n = 205). Data are presented as mean ± SEM. d Western blot analysis of PD-L1 expression in HCC827 tumor cells with UPP1 overexpression (OE) or UPP1 knockdown (SH). Representative images shown ( n = 3). e Flow cytometry analysis of PD-L1 expression on the cell surface of HCC827 tumor cells with UPP1 overexpression or UPP1 knockdown. Representative flow cytometry plots are shown ( n = 4). Data are presented as mean ± SD. Statistical analysis was conducted using the two-tailed student’s t -test. MFI, mean fluorescence intensity. f Functional enrichment analysis of six classical signaling pathways known to regulate PD-L1 in UPP1 high tumor cells. Pathways were ranked based on ssGSEA enrichment scores. g Western blot analysis of the levels of PI3K, AKT, and mTOR and phosphorylated PI3K, AKT, and mTOR in HCC827 tumor cells with UPP1 overexpression. Representative images shown ( n = 3). h Western blot analysis of the levels of phosphorylated AKT and mTOR in HCC827 tumor cells with UPP1 knockdown and after the treatment of SC79 for 48 h (20 µM). Representative images shown ( n = 3). i Flow cytometry analysis of immune effector molecules, Perforin and Granzyme B, in OT-1 CD8 + T cells after co-cultured with LLC-OVA UPP1-OE tumor cells, LLC-OVA UPP1-OENC tumor cells, and LLC-OVA UPP1-OE tumor cells with the treatment of PD-L1 antibodies (αPD-L1) or isotype control IgG. Representative flow cytometry plots shown ( n = 4). Data are presented as mean ± SD. Statistical analysis was conducted using one-way ANOVA with multiple comparisons. n denotes biologically independent samples. Source data are provided as a Source Data file.

Article Snippet: The purity of CD4 + T cells was validated using flow cytometry; Untouched CD8 + T cells were purchased from iXCells Biotechnologies (10HU-024N); CD4 + T cells or CD8 + T cells were cultured in ImmunoCult T Cell Expansion Medium (STEMCELL, 10981) supplemented with 50 U/ml IL-2 (Peprotech, 200-02-50) and ImmunoCult CD3/CD28/CD2 T cell Activator (STEMCELL, 10970).

Techniques: Two Tailed Test, Expressing, Immunohistochemistry, Western Blot, Over Expression, Knockdown, Flow Cytometry, Fluorescence, Functional Assay, Cell Culture, Control

Journal: Nature Communications

Article Title: UPP1 promotes lung adenocarcinoma progression through the induction of an immunosuppressive microenvironment

doi: 10.1038/s41467-024-45340-w

Figure Lengend Snippet: a Experimental flowchart, created with BioRender.com. b UMAP plot showing major cell populations. c tSNE plots of CD4 + T cells. From left to right: The distribution of CD4 + T cells from the UPP1-OE and UPP1-OENC groups; Expression of CD25 and FOXP3 in CD4 + T cells; The CD4 + T cells expressing both CD25 and FOXP3 are labeled as CD25 + FOXP3+ Tregs; Expression of IL-10 in CD4 + T cells. d The proportion of CD25 + FOXP3+ Tregs between the UPP1-OE and UPP1-OENC groups (left). The expression level of IL-10 in CD25 + FOXP3+ Tregs (right) ( n = 8). e tSNE plots of CD8 + T cells. From left to right: The distribution of CD8 + T cells from the UPP1-OE and UPP1-OENC groups; Expression of PD-1, LAG3, TIM3, and CTLA4 in CD8 + T cells. f Immune checkpoint expression (left) and proportion of PD1 + LAG3 + TIM3 + CTLA4+ exhausted CD8 + T cells (right) in UPP1-OE vs. UPP1-OENC groups ( n = 8). g TNFα + /IFNγ + CD8 + T cells distribution in UPP1-OE vs. UPP1-OENC groups. h Proportion of TNFα + /IFNγ + CD8 + T cells in UPP1-OE vs. UPP1-OENC groups ( n = 8). i tSNE plots of the myeloid cells. From left to right: The distribution of myeloid cells from the UPP1-OE group and UPP1-OENC group; Expression levels of PD-L1, CD163, IL-4, and IL-6 in myeloid cells. j PD-L1 expression in all CD11b+ myeloid cells between the UPP1-OE and UPP1-OENC groups (left). The proportion of CD163 + PD-L1 + M2 macrophages between the UPP1-OE and UPP1-OENC groups (right) ( n = 8). k IL-4 and IL-10 levels in CD163 + PD-L1 + M2 macrophages between the UPP1-OE and UPP1-OENC groups ( n = 8). l LLC tumor cells (UPP1-OE or UPP1-OENC) were subcutaneously injected into C57BL/6 mice. Tumors were then collected for flow cytometry analysis to compare changes in the proportion of fibroblasts ( n = 5). m Fibroblast infiltration proportion between the UPP1-OE and UPP1-OENC groups ( n = 5). n Fibroblast sorted from ( m ) for RT-qPCR detection of FAP and MMP11 expression ( n = 5). n denotes biologically independent samples. Data were represented as mean ± SD in d , f , h , j , k, and m ; mean ± SEM in n . All Statistical analysis were conducted using the two-tailed student’s t -test. Source data are provided as a Source Data file.

Article Snippet: The purity of CD4 + T cells was validated using flow cytometry; Untouched CD8 + T cells were purchased from iXCells Biotechnologies (10HU-024N); CD4 + T cells or CD8 + T cells were cultured in ImmunoCult T Cell Expansion Medium (STEMCELL, 10981) supplemented with 50 U/ml IL-2 (Peprotech, 200-02-50) and ImmunoCult CD3/CD28/CD2 T cell Activator (STEMCELL, 10970).

Techniques: Expressing, Labeling, Injection, Flow Cytometry, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Communications

Article Title: UPP1 promotes lung adenocarcinoma progression through the induction of an immunosuppressive microenvironment

doi: 10.1038/s41467-024-45340-w

Figure Lengend Snippet: a Experimental workflow, created with BioRender.com. UPP1-knockdown LLC tumor cells (sh) and control cells (shNC) were subcutaneously implanted into C57BL/6 mice with/without the treatment of with anti-PD-L1 antibodies (αPD-L1). b Tumors harvested from mice bearing LLC-UPP1-shNC tumor cells, LLC-UPP1-sh tumor cells, LLC-UPP1-shNC tumor cells treated with anti-PD-L1 antibody, and LLC-UPP1-sh tumor cells treated with anti-PD-L1 antibody ( n = 4). c Tumor growth curves ( n = 4). Statistical analysis was performed using two-way ANOVA with multiple comparisons. d Tumor weight at day 30 ( n = 4). Statistical analysis was performed using one-way ANOVA with multiple comparisons. e Flow cytometry analysis of the proportion of CD8 + T cells infiltrated in the tumors ( n = 4). Statistical analysis was performed using one-way ANOVA with multiple comparisons. f Flow cytometry analysis of immune effector molecules, Perforin and Granzyme B, in CD8 + T cells ( n = 4). Statistical analysis was performed using one-way ANOVA with multiple comparisons. g Flow cytometry analysis of immune effector molecules, TNFα+ and IFNγ + , in CD8 + T cells ( n = 4). Statistical analysis was performed using one-way ANOVA with multiple comparisons. n denotes biologically independent samples. Data are presented as mean ± SD in c – g . Source data are provided as a Source Data file.

Article Snippet: The purity of CD4 + T cells was validated using flow cytometry; Untouched CD8 + T cells were purchased from iXCells Biotechnologies (10HU-024N); CD4 + T cells or CD8 + T cells were cultured in ImmunoCult T Cell Expansion Medium (STEMCELL, 10981) supplemented with 50 U/ml IL-2 (Peprotech, 200-02-50) and ImmunoCult CD3/CD28/CD2 T cell Activator (STEMCELL, 10970).

Techniques: Knockdown, Control, Flow Cytometry