cd8 fitc  (Proteintech)

Journal: bioRxiv

Article Title: AADAT-Driven Metabolic Control of Malate and CoQ 10 Shapes Immune Evasion in Triple-Negative Breast Cancer

doi: 10.64898/2026.01.28.702389

Figure Lengend Snippet: (a) Schematic of the KP based on annotated pathways in Metacyc (PWY-6309) and KEGG (hsa00380) databases. (b) Volcano plot representing changes in the expression of KP genes in breast tumors in the NCI dataset (GSE37751) with high (n = 24) vs. low (n = 23) intra-tumoral CD8+ TILs based on median CD8+ TIL count. Red circles, upregulated genes; blue circles, downregulated genes. (c) Dot plot showing AADAT mRNA expression in breast tumors from the NCI dataset (GSE37751), divided into two subgroups of low (black, n = 23) and high (green, n = 24) CD8+ TILs divided based on the median number of intra-tumoral CD8+ TILs. Data are presented as mean ± SEM. P-value computed using Student’s t-test. (d) Dot plot showing immunohistochemical staining of AADAT scored by a pathologist in benign breast tissues (n = 23), ductal carcinoma in situ (DCIS, n = 22), invasive ductal carcinoma (IDC, n = 67), and metastatic IDC samples (n = 32) in a human breast cancer TMA (BR2082a, US Biomax). Data are presented as mean ± SEM. P-value was computed using a two-tailed Mann-Whitney test. (e) Box plot showing AADAT mRNA expression in breast tumors from the TCGA dataset across four PAM50 subtypes: Normal-like (n = 113), Luminal (n = 575), HER2-enriched (n =37), and TNBC (n = 115). Data are presented as mean ± SEM. P-value computed using Student’s t-test. (f) Dot plot showing AADAT mRNA expression in breast tumors from the METABRIC Discovery dataset across five PAM50 subtypes: Normal-like (n = 58), Luminal A (n = 466), Luminal B (n = 268), HER2-enriched (n = 87), and Basal-like (n = 118). Data are presented as mean ± SEM. P-values were computed using a two-tailed Mann-Whitney test. (g) Kaplan-Meier survival curves of disease-specific survival in breast cancer patients from the METABRIC Discovery dataset, stratified based on the median expression value of AADAT . The log-rank (Mantel-Cox) test was used to determine the P-value. (h) Kaplan-Meier survival curves of disease-specific survival in triple-negative breast cancer (TNBC) patients from the METABRIC Discovery dataset stratified by the median expression value of AADAT . The log-rank (Mantel-Cox) test was used to determine the P-value. (i) Kaplan-Meier survival curves of disease-specific survival in breast cancer patients from the SCANB dataset obtained from the ULCAN portal ( https://ualcan.path.uab.edu ). The median expression value of AADAT stratified the samples in the dataset. The log-rank (Mantel-Cox) test was used to determine the P-value. (j) Correlation plot between TIMER T cell score and AADAT mRNA expression in the TCGA breast cancer dataset (n = 112). Pearson correlation coefficient and two-tailed P-value are indicated. Dot plots showing infiltration of CD8+ T cells in TNBC tumors in the Baylor Scott and White dataset with weak (n = 12) or strong (n = 23) AADAT immunohistochemical staining. Data are presented as mean ± SEM. P-values computed using the two-tailed Mann-Whitney U test. (l) Dot plots showing the correlation between CD8+ T cells and low (n = 23) and high (n = 13), expression of AADAT in the spatial TNBC transcriptomics dataset published by Bassiouni etal (GSE210616). Data are presented as mean ± SEM. P-values computed using the student’s t-test.

Article Snippet: After 24 hours of co-culture and treatment (where applicable), the entire well of co-cultured cells was harvested for flow cytometry analysis to assess tumor cell survival and T cell populations by staining with 1:200 dilutions of CD45 (Biolegend, Cat #103155), CD8 (Cytek, Cat # 35-0081-U500), and CD3e antibodies (Biolegend, Cat # 155703).

Techniques: Expressing, Immunohistochemical staining, Staining, In Situ, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: AADAT-Driven Metabolic Control of Malate and CoQ 10 Shapes Immune Evasion in Triple-Negative Breast Cancer

doi: 10.64898/2026.01.28.702389

Figure Lengend Snippet: (a) RT-qPCR analysis for AADAT in E0771 and 4T1 cells stably transduced with control non-targeted shRNA (shNT), Aadat-specific shRNAs (shAadat), and an Aadat re-expression construct, each n=3 biological replicates. P-value computed using Student’s t-test. (b) Tumor growth analysis of wild-type C57BL/6J mice orthotopically implanted with the syngeneic E0771 cell lines described in panel a. P-values were calculated using multiple unpaired Student’s t-test. The indicated P-values are for the final study time point, i.e., Day 24. The n values are also indicated. (c) Tumor growth analysis of wild-type BALB/cJ mice orthotopically implanted with the syngeneic 4T1 cell lines stably transduced with either control non-targeted shRNA (shNT), Aadat-specific shRNAs (shAadat), and an Aadat re-expression, as described in panel a. P-values were calculated using multiple unpaired Student’s t-test. The indicated P-values are from the last study time point, i.e., Day 14. n values are shown. (d) Tumor growth analysis of C57BL/6J mice with CD8 knockout (CD8KO), orthotopically implanted with the syngeneic E0771 cell line (panel a). P-values were calculated using multiple unpaired Student’s t-tests. The indicated P values are from the final study time point, i.e., Day 19. (e) Tumor growth analysis of BALB/cJ mice with CD8 knockout (CD8KO), orthotopically implanted with the syngeneic E0771 cell line (panel a). P-values were calculated using multiple unpaired Student’s t-tests. The indicated P values are from the last study time point, i.e., Day 16. (f) RT-qPCR analysis for AADAT in E0771 cells containing doxycycline-inducible Aadat-specific shRNAs (iKD1) compared to uninduced control (control). The average of three biological replicates is shown. P-value computed using an unpaired two-tailed Student’s t-test. (g) Left panel: Representative H&E images of lung sections from wild-type C57BL/6J mice four weeks following resection of size-matched E0771 tumors expressing doxycycline-induced Aadat-specific shRNA (iKD1, n = 6, per treatment group). The primary tumors were resected at 300 mm 3 followed by induction of AADAT KD for 28 days using Doxycycline and quantification of lung metastases. Scale bar: 1 mm. Right Panel: Metastatic nodules were visually quantified in lung sections in Control and iKD1 mice. P-values computed using an unpaired two-tailed Student’s t-test.

Article Snippet: After 24 hours of co-culture and treatment (where applicable), the entire well of co-cultured cells was harvested for flow cytometry analysis to assess tumor cell survival and T cell populations by staining with 1:200 dilutions of CD45 (Biolegend, Cat #103155), CD8 (Cytek, Cat # 35-0081-U500), and CD3e antibodies (Biolegend, Cat # 155703).

Techniques: Quantitative RT-PCR, Stable Transfection, Transduction, Control, shRNA, Expressing, Construct, Knock-Out, Two Tailed Test

Journal: bioRxiv

Article Title: AADAT-Driven Metabolic Control of Malate and CoQ 10 Shapes Immune Evasion in Triple-Negative Breast Cancer

doi: 10.64898/2026.01.28.702389

Figure Lengend Snippet: (a) Analysis of clinical and transcriptomic data (GSE91061) from melanoma patients treated with immunotherapy, divided into groups with high (n = 27) or low (n = 82) expression of AADAT , revealed increased response rates (pCR + SD) in the low AADAT group (58%) vs the high AADAT group (33%). Correlation is computed by performing the Spearman correlation test, and the corresponding R and two-tailed P value are indicated. (b) Tumor growth analysis of E0771 cells expressing doxycycline-inducible Aadat-specific shRNA (iKD1) orthotopically implanted into syngeneic wild-type C57BL/6J mice. The mice were treated with one of four regimens: 1) vehicle + IgG (control), 2) doxycycline + IgG (genetic KD of AADAT ), 3) vehicle + ICB (immune checkpoint blockade therapy), and 4) doxycycline + ICB ( AADAT KD + immune checkpoint blockade therapy). Multiple unpaired Student’s t-tests determined the P-value. The indicated P values are from the final study time point, i.e., Day 33. n values are shown. (c) Tumor growth analysis of 4T1 cells expressing doxycycline-inducible Aadat-specific shRNA, orthotopically implanted into syngeneic wild-type BALB/cJ mice. The mice were treated with either four regimens: 1) vehicle + IgG (control), 2) doxycycline + IgG (genetic KD of AADAT ), 3) vehicle + ICB (immune checkpoint blockade therapy), and 4) doxycycline + ICB ( AADAT KD + immune checkpoint blockade therapy). Multiple unpaired Student’s t-tests determined the P-values. The indicated P-values are from the last study time point, i.e., Day 21. The n values are shown. (d) Representative orthotopic 4T1 tumors resected 21 days after implantation from the experiment shown in panel c. Scale bar: 1 cm. (e) Violin plot illustrating the spatial co-localization and higher interaction of CD8+ T cells and tumor cells in the AADAT iKD and control tumors shown in panels c and d. CD8+ T cells and tumor cells were identified through imaging mass cytometry analysis of paraffin-embedded 4T1 tumors (Control; n=5 mice, 16 regions of interest or ROIs; AADAT - iKD1 n=4 mice, 10 ROIs) from the experiment described in panel c. Interactions are represented in Z-scores obtained from Giotto and the significance was calculated using a two-tailed t-test.

Article Snippet: After 24 hours of co-culture and treatment (where applicable), the entire well of co-cultured cells was harvested for flow cytometry analysis to assess tumor cell survival and T cell populations by staining with 1:200 dilutions of CD45 (Biolegend, Cat #103155), CD8 (Cytek, Cat # 35-0081-U500), and CD3e antibodies (Biolegend, Cat # 155703).

Techniques: Expressing, Two Tailed Test, shRNA, Control, Imaging, Mass Cytometry

Journal: bioRxiv

Article Title: AADAT-Driven Metabolic Control of Malate and CoQ 10 Shapes Immune Evasion in Triple-Negative Breast Cancer

doi: 10.64898/2026.01.28.702389

Figure Lengend Snippet: (a) Heatmap of significantly altered metabolites (P<0.05, FDR <0.25) from an unbiased metabolomics analysis of conditioned media (CM) from E0771-ova+ TNBC cells with doxycycline-induced KD of AADAT (AADAT-iKD2) or uninduced control (Control-iKD2, n=3 biological replicates, each with three technical replicates). Shades of red and green indicate metabolites that are significantly elevated or depleted, respectively (see color scale). Metabolites belonging to the tricarboxylic acid cycle are marked with a red asterisk. CoQ 10 precursors are marked with a blue double asterisk. (b) Representative photomicrographs of a region of interest in a patient’s TNBC tumor show similar spatial clustering patterns for the CD8+ functional T cell cluster and (c) Malate, but not for (d) Fumaric acid, Phenylalanine, Leucine, Citruline, and Methylhistidine, all of which were elevated in the CM of E0771-OVA+ cells containing AADAT-KD (see ). Spatial profiles of metabolites and CD8+ functional T cells were derived from 31 TNBC tumors using two consecutive 5-micron sections of a Tissue Microarray, integrating data from imaging mass spectrometry (about 20-cell resolution) and imaging mass cytometry (single-cell resolution). Each dot represents a pixel indicating the levels (see scale bars) of metabolites or of a CD8+ functional T cell cluster. (e) Plot showing a positive correlation between average levels of functional T-cell clusters and malate. A total of 5245 aligned IMS-IMC spots from 31 TNBC patient samples were examined to explore the relationship between functional T-cells and malate levels at each location. For data visualization, spots were categorized into 10-percentile bins, and the mean values of both functional T cells and malate within each bin were plotted. A Pearson correlation coefficient was calculated, along with the P-value, using a one-tailed Student’s t-test. Results showed a significant positive correlation between functional T cells and malate across the binned spots in the TNBC tumors. (f) Same as in (e) but demonstrating a negative correlation between exhausted T cells and malate. (g) The average pixel intensity of malate, measured by imaging mass spectrometry (IMS), was assessed in each tissue core of the TNBC tissue microarray, and patients were stratified into high (n=13) and low (n=18) malate groups based on the median. An inset shows representative cores with high and low malate levels. The P-value was determined using a two-tailed unpaired Student’s t-test. (h) Kaplan-Meier plot illustrating disease-specific survival among TNBC patients, divided by median spatial malate levels. The P-value was determined using the log-rank (Mantel-Cox) test. The count of patients in each group is shown. (i) Dot plot showing Z-score normalized malate levels in conditioned media of E0771-ova+ and 4T1 cells, with two independent doxycycline-induced AADAT knockdowns (AADAT-iKD1 and AADAT-iKD2) compared to their uninduced controls (Control-iKD1 and Control-iKD2). Each group (iKD1 and iKD2) includes three biological replicates, each with three technical replicates. P values comparing induced KD to uninduced control within each group were calculated using an unpaired two-tailed Student’s t-test. The overall P value was obtained by combining the two group P values with Fisher’s method. (j) Similar to panel i, but in 4T1 cells stably transduced with either control non-targeted shRNA (shNT), Aadat-specific shRNAs (shAadat-2), or an Aadat re-expression construct (shAADAT-2+AADAT), each with three biological replicates. P values were determined using an unpaired two-tailed Student’s t-test.

Article Snippet: After 24 hours of co-culture and treatment (where applicable), the entire well of co-cultured cells was harvested for flow cytometry analysis to assess tumor cell survival and T cell populations by staining with 1:200 dilutions of CD45 (Biolegend, Cat #103155), CD8 (Cytek, Cat # 35-0081-U500), and CD3e antibodies (Biolegend, Cat # 155703).

Techniques: Control, Functional Assay, Derivative Assay, Microarray, Imaging, Mass Spectrometry, Mass Cytometry, One-tailed Test, Two Tailed Test, Stable Transfection, Transduction, shRNA, Expressing, Construct

Journal: bioRxiv

Article Title: AADAT-Driven Metabolic Control of Malate and CoQ 10 Shapes Immune Evasion in Triple-Negative Breast Cancer

doi: 10.64898/2026.01.28.702389

Figure Lengend Snippet: (a) Schematic of the co-culture showing CD8+ T cells from OT1-mice with E0771-ova+ cells that have an induced AADAT KD or uninduced controls. The uninduced controls are either treated with or without 2.5 mM malate. (b) The dot plot shows the percentage of surviving E0771-ova+ cells under conditions of induced AADAT KD or uninduced controls in a co-culture with CD8+ T cells derived from OT1-mice. The controls were either treated with or without 2.5 mM malate. Two independent inducible AADAT knockdowns (AADAT-iKD1 and AADAT-iKD2) are compared to their respective uninduced controls (Control-iKD1 and Control-iKD2). n=3 biological replicates, each with three technical replicates, were analyzed per condition. The P-value was calculated using a two-tailed unpaired Student’s t-test. (c) Volcano plot displaying differentially expressed genes (FDR<0.1, fold change ≥ 2) in CD8+ T cells from the co-culture with E0771-ova+-iKD1 cells (see panel b) and induced AADAT KD (yellow) or E0771-ova uninduced cells treated with 2.5 mM malate (red), compared to uninduced and untreated controls. Inflammatory genes are labeled. (d) Bar plot showing the results of hallmark pathways enriched by gene set enrichment analysis (GSEA, FDR<0.1) of differentially expressed genes in malate vs untreated CD8+ T cells, presented in panel b, red dots. (d) Bar plot showing common (e) Dot plot illustrating flow cytometry-derived intracellular expression of TNF-α in CD8+ T cells, treated with or without 2.5 mM malate. n=3 biological replicates, each with three technical replicates were analyzed for each condition. P-values were calculated using a two-tailed Wilcoxon matched-pairs signed rank test. (f) Same as in panel e, but for Interferon-γ. n=3 biological replicates, each with five technical replicates, were analyzed for each condition. P-values were calculated using two-tailed Wilcoxon matched-pairs signed rank test. (g) Heat map illustrating changes in mitochondrial metabolites (FDR<0.25, ≥ 1.5-fold) in CD8+ T cells treated with or without 2.5 mM malate. Three biological replicates, each with two technical replicates, were analyzed. Shades of yellow and blue indicate increased or decreased metabolite levels (see color scale). C1-C3: Control; M1-M3: Malate-treated. (h) NAD: NADH ratio in CD8+ T cells treated with or without 2.5 mM malate. Ten biological replicates were analyzed. P-values were calculated using a two-tailed unpaired Student’s t-test. (i) Reactive oxygen species (ROS) analysis with CD8+ T-cells divided into four groups: Pyocyanin (positive control) treated, N-acetylcysteine (negative control) treated, Untreated, and 2.5 mM Malate treated. n=3 biological replicates, each with five technical replicates were analyzed for each condition. P-values were calculated using two-tailed Wilcoxon matched-pairs signed rank test.

Article Snippet: After 24 hours of co-culture and treatment (where applicable), the entire well of co-cultured cells was harvested for flow cytometry analysis to assess tumor cell survival and T cell populations by staining with 1:200 dilutions of CD45 (Biolegend, Cat #103155), CD8 (Cytek, Cat # 35-0081-U500), and CD3e antibodies (Biolegend, Cat # 155703).

Techniques: Co-Culture Assay, Derivative Assay, Control, Two Tailed Test, Labeling, Flow Cytometry, Expressing, Positive Control, Negative Control

Journal: Advanced Science

Article Title: Engineered Bacteria‐Vesicle Delivered Lactate Reprogramming Boosts Tumor Radiosensitivity

doi: 10.1002/advs.202524303

Figure Lengend Snippet: In vivo immune remodeling and tumor microenvironment modulation. (a) Schematic diagram of tumor microenvironment remodeling post‐treatment. Created with BioRender. (b–d) Representative IHC staining of Pan‐Kla, HIF1α, and IFN‐γ in tumor sections, scale bar = 50 µm. (e,f) Representative flow cytometry images of CD8 + T cells (CD45 + CD3 + CD8 + ) and M1 macrophages (CD11b + F4/80 + CD86 + ) in tumor tissues. (g) Representative flow cytometry images of DCs (CD11c + CD80 + CD86 + ) in mesenteric lymph nodes. (h–j) Quantitative immune cell profiling: (h) CD8 + T cell infiltration, (i) M1 macrophage polarization, and (j) DC maturation in mesenteric lymph nodes ( n = 6). (k) Quantification of IFN‐γ immunohistochemical staining in mice from different treatment groups ( n = 4). All data represent mean ± SD. Statistical significance was determined by one‐way ANOVA with Tukey's multiple‐comparisons test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns. not significant).

Article Snippet: For flow cytometry, the following anti‐mouse antibodies were used: CD3‐FITC (Biolegend, 100202), CD4‐APC (Biolegend, B201509), CD8‐PE (Biolegend, 100734), CD11c‐FITC (Elabscience, N418), CD86‐APC (Biolegend, 105008), CD80‐PE (Biolegend, 104708), CD11b‐PerCP (Elabscience, E‐AB‐F1081F), and F4/80‐FITC (Elabscience, E‐AB‐F0995C), CD8 alpha (RPA‐T8) Mouse Monoclonal Antibody FITC Conjugate (Cell signaling, 55397).

Techniques: In Vivo, Immunohistochemistry, Flow Cytometry, Immunohistochemical staining, Staining