4t1 atcc crl 2539  (ATCC)

Journal: PLOS ONE

Article Title: A novel TREX1 inhibitor, VB-85680, upregulates cellular interferon responses

doi: 10.1371/journal.pone.0305962

Figure Lengend Snippet: A) TREX1 Western blot demonstrating overexpression of both wild-type mouse TREX1 and mouse TREX1 carrying the D18N inactivating mutation. Endogenous mouse TREX1 present in a J774 cellular lysate is shown as reference to confirm the size of the upper band as full-length mouse TREX1. α-tubulin was used as a loading control. B–D) Exonuclease assays performed with HEK293T mouse TREX1 overexpression or empty vector lysates demonstrate the effects of VB-85680 and VB-85662 on B) wild-type mouse TREX1 (5 ng/μL total protein), C) Catalytically inactive mouse TREX1 D18N (100 ng/μL total protein) and, D) endogenous exonuclease activity (100 ng/μL total protein). E) Western blot for endogenous levels of cGAS,STING and TREX1 in both 4T1 and THP1-dual cells. F) Endogenous exonuclease activity in 4T1 cell lysates is inhibited by VB-85680, but not by VB-85662. Compounds were titrated in the presence of 6 μg 4T1 cytosolic lysate using the 3’ to 5’ Exonuclease Activity Assay from BioVision. G) Inhibition of endogenous human TREX1 exonuclease activity by VB-85680 in THP1-Dual™ and THP1-Dual™ TREX1 knockout cell lysates. The exonuclease assay was carried out as described in 1E . Error bars for all experiments represent +/- SD.

Article Snippet: Briefly, THP1-Dual™ (Invivogen thpd-nfis), THP1-Dual™ TREX1 KO (Invivogen thpd-nfis) or 4T1 (ATCC CRL-2539) cells were suspended at a density of 1 million cells/mL in the lysis buffer provided.

Techniques: Western Blot, Over Expression, Mutagenesis, Control, Plasmid Preparation, Activity Assay, Inhibition, Knock-Out

Journal: Nature Communications

Article Title: Blocker-SELEX: a structure-guided strategy for developing inhibitory aptamers disrupting undruggable transcription factor interactions

doi: 10.1038/s41467-024-51197-w

Figure Lengend Snippet: a CoIP assays of WMiApt-1 in blocking the interaction between WDR5 and MYC (biological replicates n = 2). Source data are provided as a Source Data file. b Protein levels of MYC and WDR5 of three human-derived cell lines. c Anti-proliferation effects of WMiApt-1 in three human cell lines The result is determined from three biological replicates, each containing three technical replicates (biological replicates n = 3, mean ± SD, Two-sided Student’s t -test). The 95% confidence intervalare −56.79 to −36.85, −23.20 to −2.945 and −8.267 to −0.7118. Source data are provided as a Source Data file. Source data are provided as a Source Data file. d Schematic diagram illustrating the anti-tumorigenesis experiments in-vivo. Icons from Canva. e Tumor volumes of 4T1 cells treated with WMiApt-1 or control on the fifth day post-implantation (biological replicates n = 5, mean ± SD, Two-sided Student’s t -test). The 95% confidence interval is −8.267 to −0.7118. Source data are provided as a Source Data file.

Article Snippet: MDA-MB-468 (HTB-132), HEK293T (ACS-4500), SKNBE2 (CRL-2271), 4T1 (CRL-2539) cell lines were purchased from ATCC.

Techniques: Blocking Assay, Derivative Assay, In Vivo, Control

Journal: Nature Communications

Article Title: Reprogramming the tumor immune microenvironment using engineered dual-drug loaded Salmonella

doi: 10.1038/s41467-024-50950-5

Figure Lengend Snippet: a Experimental scheme. Briefly, 4T1 cells were implanted into the mammary fat pad of BALB/c mice. When tumors reached approximately 140 mm 3 , mice received bacteria through i.v injection (day 0). Doxy (+) and CSF-1R antibody (αCSF-1R) were administered as shown. Pimary tumors and metastatic lungs were collected on day 15 and 35, respectively ( b , c ) and flow cytometry was performed on day 9 ( d , e ). b Average growth curves of 4T1 primary tumors ( n = 6 mice examined from two independent experimental replicates; * P = 0.0144, **** P < 0.0001; two-way ANOVA with Tukey’s multiple comparisons test). c Individual images (left) and metastatic nodule count (right) of lungs ( n = 6 exami n ed from two independent experimental replicates; **** P < 0.0001; ns not significant; unpaired two-tailed t -test). d Flow cytome t ry gating of M1 (F4/80 + CD86 + ) and M2 (F4/80 + CD206 + ) macrophages . e Ratio of M1/M2 macrophages in lungs (left) and tumors (right) ( n = 6 mice examined from two independent experimental replicates; **** P < 0.0001; ns, not significant; unpaired two-tailed t -test). f In vivo and ex vivo bioluminescent imaging of MDA-MB-231-FLuc-GFP lung metastasis. Left, images of the mice on day 0 (pre-treatment) and on day 14 (post-treatment) and the excised lungs on day 14; right, quantitation of bioluminescence levels in the lungs. g Frequency of macrophages phagocytosing tumor cells (F4/80 + GFP + ) in the metastatic lungs of ( f ) ( n = 3 mice/group, from one experiment, unpaired two-tailed t -test). h Experimental scheme. i Average growth curves of subcutaneous tumors ( n = 7 mice examined from two independent experimental replicates; **** P < 0.0001; two-way ANOVA with Tukey’s multiple comparisons test). j Kaplan-Meier survival curves ( n = 7 mice examined from two independent experimental replicates; **** P < 0.0001; Log-rank (Mantel-Cox) test). k Metastatic scores of livers. Metastatic nodules on the liver surface were counted at the experimental end point. The scores were classified as follows: 0, 0%; 1, <20%; 2, 20%–50%; 3, >50% ( n = 7 mice examined from two independent experimental replicates; **** P < 0.0001; unpaired two-tailed t -test).

Article Snippet: CT26 (mouse colon carcinoma, CRL-2638), 4T1 (mouse breast cancer, CRL-2539), and HEK293T (human epithelial cell, CRL-3216) cell lines were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: Bacteria, Injection, Flow Cytometry, Two Tailed Test, In Vivo, Ex Vivo, Imaging, Quantitation Assay

Journal: Cancers

Article Title: Evaluation of the Mammalian Aquaporin Inhibitors Auphen and Z433927330 in Treating Breast Cancer

doi: 10.3390/cancers16152714

Figure Lengend Snippet: Auphen treatment of metastatic breast cancer cells increases overall survival in vivo. ( A ) Schematic of orthotopic injection of 4T1 cells in NOD SCID mice treated with either vehicle ( n = 15) or Auphen ( n = 15). ( B ) Growth of tumors in ( A ). ( C ) Kaplan–Meier plot showing overall survival (OS). p -values for OS were determined using the log-rank (Mantel–Cox) test. ( D ) Quantification of median lung metastasis using Welch’s t -test. ( E ) Schematic of orthotopic injection of 4T1 cells in BALB/cJ mice treated with either vehicle ( n = 14) or Auphen ( n = 14). ( F ) Tumor burden of ( E ). ( G ) Kaplan–Meier plot showing overall survival (OS) of ( E ). p -values for OS were determined using the log-rank (Mantel–Cox) test. ( H ) Quantification of median lung metastasis was analyzed using Goodman and Kruskal’s gamma test.

Article Snippet: Mouse 4T1 (CRL-2539) and human AU565 (CRL-2351), BT474 (HTB-20), MCF-7, and MDA-MB-231 cells were purchased from ATCC.

Techniques: In Vivo, Injection

Journal: Cancers

Article Title: Evaluation of the Mammalian Aquaporin Inhibitors Auphen and Z433927330 in Treating Breast Cancer

doi: 10.3390/cancers16152714

Figure Lengend Snippet: Z433927330 treatment of metastatic mammary tumors in vivo. ( A ) Schematic of orthotopic injection of 4T1 cells in syngeneic BALB/cJ mice treated with either vehicle ( n = 4, weekly and biweekly) or Z433927330 ( n = 4, weekly and biweekly). ( B ) Tumor growth curves for biweekly treatment. ( C ) Quantification of lung metastasis using Fisher’s exact test. ( D ) Kaplan–Meier plot showing overall survival for biweekly treatment. p -values for OS were determined using the log-rank (Mantel–Cox) test. ( E ) Tumor growth curves for weekly treatment. ( F ) Quantification of lung metastasis using Goodman and Kruskal’s gamma. ( G ) Kaplan–Meier plot showing overall survival for weekly treatment. p -values for OS were determined using the log-rank (Mantel–Cox) test.

Article Snippet: Mouse 4T1 (CRL-2539) and human AU565 (CRL-2351), BT474 (HTB-20), MCF-7, and MDA-MB-231 cells were purchased from ATCC.

Techniques: In Vivo, Injection

Journal: Molecular Therapy. Nucleic Acids

Article Title: Combining mRNA with PBS and calcium ions improves the efficiency of the transfection of mRNA into tumors

doi: 10.1016/j.omtn.2024.102273

Figure Lengend Snippet: The evaluation of several materials to introduce mRNA into tumors (A) Schema of the mRNA coding luciferase 2. Luciferase 2 mRNA is generated with Cap-1 and 1-methyl-pseudouridine through in vitro transcription. PolyA is encoded in the template plasmid. (B) Electrophoresis of the mRNA generated through in vitro transcription using a Tape station. (C) Dot plot showing the region of interest (ROI) values measured using an IVIS. The number in parentheses indicates the number of tumors. We calculated p values using the Wilcoxon test. Right images show luciferase signals by the intra-tumoral injection of mRNA with MilliQ, PBS, sucrose, and in vivo -jetRNA (invivoJET). (D) Representative images of mice intratumorally injected with mRNA with COMIRNATY-composed LNP in CT26 tumor-bearing BALB/c and B16F10 tumor-bearing C57BL/6 mice. White contour indicates the location of the tumors. (E–G) Dot plot showing the ROI values in MC38 (E) or LL2 (F) tumor-bearing C57BL/6 and 4T1 (G) tumor-bearing BALB/c mice. (C, E–G) The dot plots have mean lines, mean error bars, and SD lines.

Article Snippet: B16F10 (CRL-6475), LL/2 (CRL-1642), 4T1 (CRL-2539), and CT26 (CRL-2638) cells were purchased from the American Type Culture Collection.

Techniques: Introduce, Luciferase, Generated, In Vitro, Plasmid Preparation, Electrophoresis, Injection, In Vivo

Journal: Nature Communications

Article Title: Loss of tumor suppressors promotes inflammatory tumor microenvironment and enhances LAG3+T cell mediated immune suppression

doi: 10.1038/s41467-024-50262-8

Figure Lengend Snippet: A Schematic loss-of-function screening for TS genes in 4T1 mammary tumor metastasis (created with BioRender.com). B Lung nodule counts at 3 weeks (left) or 4 weeks (right) after tumor cell injection and C Lung nodule counts with different sizes from immune-deficient mice received tumor cell injection. The number and size of tumor nodules on lung surfaces were counted under a dissecting microscope. n = 8 per group. D Representation of sgRNA library at different stages of tumor growth and metastasis. Number of sgRNA species in cells before transplantation, different stages of primary tumor, lymph node, and lung during tumor evolution. E Pie charts for the most abundant sgRNAs in the lung after 2, 3, and 4-week sgRNA library-mediated cell injection. F Enriched sgRNAs in the lung nodules at 3 and 4 weeks after cell transplantation. Individual tumor nodules were taken out from the lungs, PCR amplified the sgRNA sequence, and examined through Sanger sequencing. G Dynamic evolution of sgRNAs indicated during tumor growth and metastasis. All sgRNAs with ≥2% of total reads are plotted individually. The remaining lung indicates most of the nodules were picked out before the evaluation. n = 8 per group. All bar graphs show mean ± s.d. Statistical significance was determined by two-tailed Student’s t -test for ( C ).

Article Snippet: 4T1 (Cat#, CRL-2539) and EMT6 (Cat#, CRL-2755) cell lines were obtained from the American Type Culture Collection, and TSAE1 cell line was kindly provided by Dr. Lalage M. Wakefield from the lab of cancer biology and genetics, National Cancer Institute.

Techniques: Injection, Microscopy, Transplantation Assay, Amplification, Sequencing, Two Tailed Test

Journal: Nature Communications

Article Title: Loss of tumor suppressors promotes inflammatory tumor microenvironment and enhances LAG3+T cell mediated immune suppression

doi: 10.1038/s41467-024-50262-8

Figure Lengend Snippet: A Western blot evaluation of individual sgRNAs induced Nf1 , Tsc1 , and Tgfbr2 deletion , as well as Nf2 KO. 3–4 sgRNAs were selected for each gene from the sgRNA library and used for validation. The Western blots were repeated for the cells with most significant KO for each TS gene and showed similar results. B Representative H&E staining of the lungs and number of metastatic nodules. Lung surface metastasis normalized to tumor mass of nude mice. n = 7 per group. C Primary tumor growth in nude mice transplanted with 4T1-C1 cells with sgRNA knockdown of Nf1 , Tsc1 , or Tgfbr2 ( n = 9 per group). D , E Western blot for shRNA knockdown of Nf1 , Tsc1 , or Tgfbr2 ( D ), and representative Indian ink staining of the lungs ( E left) and metastatic nodule counts (E right) ( n = 10 mice each group). F Number of lung metastatic nodules (normalized to primary tumor weight) comparing nude and Cas9 transgenic mice that received MFP injection of tumor cells with sgRNA mediated Nf1 , Tsc1 , or Tgfbr2 deletion. G – I TSAE1+mHer2 mouse model: Cas9 transgenic mice received TVI of TSAE1 tumor cells with sgRNA mediated Nf1 , Tsc1 , or Tgfbr2 deletion: Western for Her2 overexpression in TSAE1 tumor cells ( G ); Western for sgRNA KO of Nf1 , Tsc1 , or Tgfbr2 ( H ); metastatic nodule counts from lungs ( n = 10 mice each group) I . Statistical significance was determined by one-way ANOVA followed by Sidak’s test for ( B ); two-tailed Student’s t -test for ( E ), ( F ), and ( I ). All graphs show mean ± s.d. * p < 0.05; ** p < 0.01; *** p < 0.001. Exact p -values are provided in a source data file for ( F ).

Article Snippet: 4T1 (Cat#, CRL-2539) and EMT6 (Cat#, CRL-2755) cell lines were obtained from the American Type Culture Collection, and TSAE1 cell line was kindly provided by Dr. Lalage M. Wakefield from the lab of cancer biology and genetics, National Cancer Institute.

Techniques: Western Blot, Staining, Knockdown, shRNA, Transgenic Assay, Injection, Over Expression, Two Tailed Test

Journal: Nature Communications

Article Title: Loss of tumor suppressors promotes inflammatory tumor microenvironment and enhances LAG3+T cell mediated immune suppression

doi: 10.1038/s41467-024-50262-8

Figure Lengend Snippet: A , B Western blot of the protein extractions from mouse primary tumor and lung metastasis in 4T1 and EMT6 syngeneic orthotopic mouse models. The Western blots were repeated for both models with additional samples and showed similar results. C Immunofluorescence staining of NF1, TSC1, and TβRII in the primary tumor and lung metastasis collected at 5 weeks after 4T1 tumor cell injection. Representative images are shown. Quantitative data on fluorescence intensity are on the right. Statistical significance was determined by two-tailed Student’s t -test for ( C ). All graphs show mean ± s.d.

Article Snippet: 4T1 (Cat#, CRL-2539) and EMT6 (Cat#, CRL-2755) cell lines were obtained from the American Type Culture Collection, and TSAE1 cell line was kindly provided by Dr. Lalage M. Wakefield from the lab of cancer biology and genetics, National Cancer Institute.

Techniques: Western Blot, Immunofluorescence, Staining, Injection, Fluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: Loss of tumor suppressors promotes inflammatory tumor microenvironment and enhances LAG3+T cell mediated immune suppression

doi: 10.1038/s41467-024-50262-8

Figure Lengend Snippet: A schematic analysis for ATAC and RNA-seq datasets. B RNA-seq Venn diagram showing overlapped genes from in vitro cultured 4T1 cells with sgRNA mediated deletion of Nf1, Tsc1, or Tgfbr2 compared with control 4T1-C1 cells. Cut-off: FC > 1.5, P < 0.05. C ATAC-seq Venn diagram (left) and heatmap (right) showing common differential peaks for all three TS KO vs the control cells. Cut-off: Differential ATAC peaks p < 0.01, FC < −2 and >2. D Increased ATAC peaks for IL6, JAK3 and decreased ATAC peaks for CCL2 that are common for all three TS KO cells, Gapdh as control. E Relative expression ( RT-PCR) of IL6, JAK3, IDO1, and CCL2 comparing sgNf1, sgTsc1 or sgTgfbr2 vs control ( n = 4). (F) IL6 ( n = 3) and IDO1 ( n = 3) ELISA (left two panels), JAK3 Western, and CCL2 Cytokine Array and quantitative data (right two panels, IL-23 as control) from sgNf1, sgTsc1 or sgTgfbr2 compared with control. G Western blots of pSTAT3, pSTAT6, pJAK3 and JAK3 comparing sgNf1, sgTsc1 or sgTgfbr2 4T1 cells with C1 control. H RT-PCR of IL6 or IDO1 of cells treated with a pan JAK or a JAK3 specific inhibitor or IL6-neuAb ( n = 3). All graphs show mean ± s.d. Average of each biological replicate was plotted, and statistical significance was determined by two-tailed Student’s t -test for ( E ), ( F ), and ( H ). * p < 0.05; ** p < 0.01. Exac t p -values for ( E ), ( F ), and ( H ) are provided in a source data file.

Article Snippet: 4T1 (Cat#, CRL-2539) and EMT6 (Cat#, CRL-2755) cell lines were obtained from the American Type Culture Collection, and TSAE1 cell line was kindly provided by Dr. Lalage M. Wakefield from the lab of cancer biology and genetics, National Cancer Institute.

Techniques: RNA Sequencing Assay, In Vitro, Cell Culture, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Neutralizing Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Loss of tumor suppressors promotes inflammatory tumor microenvironment and enhances LAG3+T cell mediated immune suppression

doi: 10.1038/s41467-024-50262-8

Figure Lengend Snippet: A Venn diagram for differential gene expression comparing the primary tumors with sgNf1, sgTsc1, or sgTgfbr2 with controls in Cas9 transgenic mice. Cut-off: FC > 2, P < 0.01. B Heatmap of differentially regulated genes shared by all three primary tumors with sgNf1, sgTsc1, or sgTgfbr2 compared with control tumors. C IPA analysis of 150 genes that are shared among the NF1, TSC1, or TβRII deficient 4T1 tumor cells. D Heat map for major altered immune modulators in 4T1 tumor cells with NF1, TSC1, or TβRII deficiency. The gene names are listed on the right. Red color indicates genes shared among NF1, TSC1, or TβRII deficient cells. E Flow cytometry of various myeloid subsets from primary tumors with NF1, TSC1, and TβRII deficiency vs C1 control, showing % of MDSCs, monocytes, macrophages, cDC1, and cDC2. F Metastatic nodule counts (normalized to tumor weight) upon depletion of Treg or Ly6G cells comparing the Cas9 transgenic vs nude mice that bear TSC1 deficient tumors. Graph shows mean ± s.d. G CODEX imaging of immune cells from primary tumors with NF1, TSC1, or TβRII deficiency. The blue, orange, and pink arrows show LY6G+ neutrophils, CD25+ Tregs, and PD1+ CD8+ T cells, respectively. H CYTEK analysis of primary tumors with NF1, TSC1, or TβRII deficiency. Upper: percentage of CTL, Treg, and Th2; Lower: percentage of PD-1+ in CD8 and CD4 T cells as well as percentage of LAG3+ in CD8, CD44CD8, and Treg cells. Graphs show mean ± s.d. I % of LAG3+ T cells in tumors ( n = 4) treated with Tofacitinib. J Metastatic nodule counts (normalized to tumor weight) and tumor weight with Tofacitinib treatment. K Schematic hypothesis showing how NF1, TSC1, or TβRII deficiency leads to altered inflammatory and immune suppressive microenvironment. All graphs show mean ± s.d. Statistical significance was determined by Wald test for ( A ); two-tailed Student’s t -test for ( F ) and ( H ); one-way ANOVA followed by Sidak’s test for ( I ) and ( J ). * p < 0.05; ** p < 0.01; *** p < 0.001. Exact p -values for ( H ) are provided in a source data file.

Article Snippet: 4T1 (Cat#, CRL-2539) and EMT6 (Cat#, CRL-2755) cell lines were obtained from the American Type Culture Collection, and TSAE1 cell line was kindly provided by Dr. Lalage M. Wakefield from the lab of cancer biology and genetics, National Cancer Institute.

Techniques: Expressing, Transgenic Assay, Control, Flow Cytometry, Imaging, Two Tailed Test

Journal: Nature Communications

Article Title: Loss of tumor suppressors promotes inflammatory tumor microenvironment and enhances LAG3+T cell mediated immune suppression

doi: 10.1038/s41467-024-50262-8

Figure Lengend Snippet: A Heatmap for the expression of NF1, TSC1, and TβRII in breast cancer subtypes (TCGA dataset). B Kaplan–Myer survival curve of breast cancer patients with the expression levels of NF1, TSC1, or TβRII in basal subtype breast cancer. C TS-Imm signature gene list, and the predicted Kaplan–Myer survival curve of all and basal, Her2+, Lum A/B subtype breast cancer patients. D Heatmap for the correlation of TS expression with LAG3 (left) and in subtypes of breast cancer (right), TCGA dataset. E upper: Schematic of combination treatment of anti-LAG3, anti-PD-L1, and Pac in 4T1 preclinical mouse model. lower: metastatic nodule counts normalized to tumor weight. F CyTEK analysis of primary tumors with NF1, TSC1, or TβRII deficiency from mice that were treated with anti-LAG3, anti-PD-L1, and Pac. Upper: percentage of CD4 and Treg cells; Lower: percentage of CD8, LAG3+ CD8, and PD-1+ CD8 T cells ( n = 4). G Number of metastatic nodules per tumor weight in mice treated with anti-LAG3, IDO1 inhibitor, and Pac (left); LAG3+ CD8 and LAG3+ CD4 T cells from anti-LAG3 antibody, IDO1 inhibitor and Pac treatment ( n = 4) (middle and right panels). Statistical significance was determined by logrank test for ( B ) and ( C ); one-way ANOVA followed by Sidak’s test for E ; two-tailed Student t -test for ( F ) and ( G ). All graphs show mean ± s.d.

Article Snippet: 4T1 (Cat#, CRL-2539) and EMT6 (Cat#, CRL-2755) cell lines were obtained from the American Type Culture Collection, and TSAE1 cell line was kindly provided by Dr. Lalage M. Wakefield from the lab of cancer biology and genetics, National Cancer Institute.

Techniques: Expressing, Two Tailed Test