Journal: Clinical Cancer Research

Article Title: The Potential of the Gut Microbiota and Butyrate to Enhance CAR T-cell Therapy in Non-Hodgkin Lymphoma

doi: 10.1158/1078-0432.CCR-25-1676

Figure Lengend Snippet: The gut microbiota prior to CAR T-cell treatment differs between responders and nonresponders and constitutes a prognostic factor. A, PFS curves of patients classified based on high or low indices of bacterial richness, α-diversity, and evenness of the gut microbiota ( n = 57). According to maximally selected rank statistics, the cutoff points for categorization were calculated at 150, 3.487, and 0.641 for the richness, α-diversity, and evenness indices, respectively. B, Taxonomic comparison of gut microbiota families with significantly different relative abundance before CAR T-cell treatment between responders and nonresponders ( n = 57). C, Taxonomic comparison of gut microbiota genera with significantly different relative abundance before CAR T-cell treatment between responders and nonresponders ( n = 57). The analysis shows taxa with differential abundance between responders (green) and nonresponders (red) based on log 2 fold change values. Log 2 fold change was calculated as follows: log 2 (mean relative abundance in responders/mean relative abundance in nonresponders). Therefore, positive values indicate higher abundance in responders, whereas negative values indicate higher abundance in nonresponders. D, Schematic representation of fecal microbiota transplantation (FMT) validation in an A20 immunocompetent model. Mice were subjected to endogenous microbiota depletion by administering a cocktail of antibiotics for 3 days. Subsequently, FMT was performed on four alternate days. Fourteen days after the last FMT, A20 tumor cells were injected, followed by treatment with murine CD19 CAR T cells 14 days later. E, Survival curve of the experiment with A20 mice transplanted with microbiota from responder ( n = 7) and nonresponder ( n = 8) patients subsequently treated with murine CD19 CAR T cells, in comparison with A20 control mice injected with saline ( n = 9) or mock-transduced T cells ( n = 9). ASV, amplicon sequence variant; CP, cyclophosphamide.

Article Snippet: Two weeks after this, 5 × 10 5 murine aggressive lymphoma A20 cells (RRID:CVCL_1940; cat. #TIB-208, ATCC) were intravenously injected into the tail vein, and 1 × 10 6 murine CD19 CAR T cells ( ) were intravenously injected into the tail vein 14 days later.

Techniques: Comparison, Transplantation Assay, Biomarker Discovery, Injection, Control, Saline, Amplification, Sequencing, Variant Assay

Journal: bioRxiv

Article Title: Indomethacin exerts both cyclooxygenase inhibition-dependent and independent mechanisms to enhance chemo-immunotherapy in mice

doi: 10.64898/2026.01.07.698231

Figure Lengend Snippet: Indo enhances chemo-immunotherapy through both COX-dependent and COX-independent mechanisms. (A) Western blot analysis showing COX-2 expression in the indicated mouse tumor cell lines. β-actin was used as a loading control. (B) PGE₂ concentrations in tumor cell culture supernatants. Tumor cells were seeded in 6-well plates (0.5 × 10⁶ cells per well), and supernatants were collected at confluence. PGE₂ levels were quantified by LC-MS. Data are shown as mean ± SEM of triplicate samples. (C) Quantification of PGE₂ in tumor tissues. BALB/c mice were implanted s.c. with CT26 or A20 tumor cells. Tumor tissues were resected at the sizes of 100-200 mm 2 and processed for PGE₂ quantification by LC-MS. Results were normalized to tumor tissue weight and shown as mean ± SEM of triplicate samples. (D) CTX+Indo augments the efficacy of aPD1 treatment in the A20 tumor model. The schema depicts the timeline of the experimental procedures. Mice with established A20 tumors (80-100mm 2 ) were randomly assigned to groups to receive the indicated treatments. Tumor growth curves are shown, with the number of mice per group indicated. Data shown are pooled from two independent experiments. Mouse survival is summarized in the Kaplan-Meier plot (E). (F) CT26.COXKO cells do not produce measurable PGE₂. Supernatants from CT26.COXKO cell culture were collected for PGE₂ quantification by LC-MS. Cell cultures from untreated or Indo-treated wild-type CT26 cells were included as controls. Results are shown as mean ± SEM of triplicate samples. (G) PGE₂ is not detectable in CT26.COXKO tumor tissues regardless of treatment. BALB/c mice with established wild-type CT26 or CT26.COXKO tumors (60-90mm 2 ) were randomly assigned to three groups to receive no treatment, CTX alone, or CTX+Indo. 7 days after CTX, tumors were harvested and processed for PGE₂ quantification by LC-MS. Results were normalized to tumor tissue weight and shown as mean ± SEM of triplicate samples. (H) Waterfall plots summarizing the responses of CT26.COXKO tumor response to the indicated treatments. As depicted in the schema, mice with established CT26.COXKO tumors (60-90mm 2 ) were randomly assigned to groups to receive the indicated treatments. Tumor size changes from treatment initiation to endpoint were normalized to the initial tumor sizes for each mouse and used to generate the waterfall plots. (I) Indo reduces the level of RAS-GTP in CT26 cells. CT26 cells were treated with DMSO or Indo (10uM) for 16 hours before being harvested for RAS pull-down assays. Similar assays were conducted for CT26.COXKO cells (J) and A20 cells (K). Representative Western blots for RAS-GTP and total RAS are shown. Normalized RAS-GTP values were calculated as the ratio of RAS-GTP to total RAS in Indo-treated samples divided by the corresponding ratio in DMSO-treated samples. Data are summarized as mean ± SEM from at least three biological replicates per condition. Statistics: (B, F, G, I, J, K), one-way ANOVA; (C), student t- test; (E) Log-rank (Mantel-Cox) test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.

Article Snippet: A20, 4T1, MC38, and CT26 mouse tumor cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Expressing, Control, Cell Culture, Liquid Chromatography with Mass Spectroscopy

Journal: Immunotherapy Advances

Article Title: Targeting intratumoral regulatory T cells by CD137 aptamer-shRNA chimeras

doi: 10.1093/immadv/ltaf037

Figure Lengend Snippet: Selective, dose-dependent binding and uptake of murine CD137 aptamer by CD137-expressing cells. A total of 2 × 10 5 A20 or A20-CD137 cells prefixed with 1% paraformaldehyde were treated with fluorescein maleimide (FAM)-labeled murine CD137 aptamer at different concentrations (50, 100, and 500 nM). (a) The size of murine aptamer (222 nt) and the successful labeling of the aptamer with fluorescein maleimide (FAM) were detected using agarose gel electrophoresis. (b) CD137 expression on A20 and A20-CD137 cells. (c) Histograms showing the binding of CD137 aptamer to A20 (upper panel) or A20-CD137 (bottom panel) cells when given at different concentrations. (d) Comparisons of CD137 aptamer binding across different concentrations in A20 or A20-CD137 cells. (e) Comparisons of the % population between A20 and A20-CD137 cells that had bound to the CD137 aptamer at a given concentration. A total of 5 × 10 5 CD137-expressing (A20-CD137 or B16-CD137) or control (A20 or B16) cell lines were incubated with murine CD137 aptamer (0.5, 1, or 2 μg) for 2 h at 37°C. Comparisons between the amount of CD137 aptamer internalized by (f) A20 and A20-CD137 or (g) B16 and B16-CD137 cells. Comparisons of CD137 aptamer uptake when given at different amounts for (h) A20-CD137 and (i) B16-CD137 cells. The extent of CD137 aptamer internalization was expressed as fold change relative to control cells treated with 0.5 μg aptamer, or as absolute copy number per 1 μg total RNA. Dashed lines at a ratio of 1 represent the normalization reference. Data are shown as means ± SEM. Numbers above the brackets indicate P -values. * P < .05, ** P < .01, and *** P < .001 using unpaired Students’ t -test for (e), (f), and (g), while using one-way ANOVA with Bonferroni’s multiple comparison test for (d), (h), and (i).

Article Snippet: Murine B lymphoma cell line A20, melanoma cell line B16, and hepatoma cell line Hepa1-6 were purchased from American Type Cell Culture (ATCC; Manassas, VA, USA).

Techniques: Binding Assay, Expressing, Labeling, Agarose Gel Electrophoresis, Concentration Assay, Control, Incubation, Comparison

Journal: Immunotherapy Advances

Article Title: Targeting intratumoral regulatory T cells by CD137 aptamer-shRNA chimeras

doi: 10.1093/immadv/ltaf037

Figure Lengend Snippet: Murine CD137 aptamer blocks trogocytosis induced by intercellular CD137-CD137L interaction. CD137L-expressing RAW264.7 were incubated with A20 or A20-CD137 cells at a 1:1 ratio for 1 h at 37°C. During this process, anti-CD137 blocking antibody (clone: 17B5) or CD137 aptamer was added. Changes in expressions of CD137 and CD137L on (a) RAW264.7 and A20 cells or (b) RAW264.7 and A20-CD137 cells during coculture. The number in each histogram plot represents the percentage of positive cells. Each symbol represents an independent experiment. Data are presented as means ± SEM. *** P < .001 using one-way ANOVA with Bonferroni’s multiple comparison test. MFI, mean fluorescence intensity.

Article Snippet: Murine B lymphoma cell line A20, melanoma cell line B16, and hepatoma cell line Hepa1-6 were purchased from American Type Cell Culture (ATCC; Manassas, VA, USA).

Techniques: Expressing, Incubation, Blocking Assay, Comparison, Fluorescence