4t1 cat crl 2539  (ATCC)

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

Journal: Nature Communications

Article Title: Cholesterol-modified sphingomyelin chimeric lipid bilayer for improved therapeutic delivery

doi: 10.1038/s41467-024-46331-7

Figure Lengend Snippet: a A table delineating the physicochemical characterizations of DOX/Lipo with eq. 35 mol % Chol (eq. 64.4 mol % Chol for DOX/Lipo-PChcPC) and 5 mol % DSPE-PEG 2K (n = 3 independent experiments). b – f anti-TNBC effects in metastatic orthotopic 4T1-Luc2 tumor mouse model. A total of 2 × 10 5 cells were injected into the 4 th mammary fat pad of BABL/c mice (n = 5 mice) . On day 15, the mice with primary tumors ~200 mm 3 ( b ) received an i.v. administration of various DOX/Lipo or Doxil at 15 mg DOX/kg. c Average tumor growth curves measured by a digital caliper. d mice BLI on day 15, 20, 29, and 35 by Lago optical imaging. Ex vivo lung metastasis BLI from all 5 mice in each group ( e ) and tumor-bearing mice images ( f ) were taken on day 35. Data in a (right portion), c (n = 5 mice) are expressed as mean ± s.d. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test; survival curves were compared using the log-rank Mantel–Cox test. Source data are provided as a Source Data file.

Article Snippet: CT26 (Cat. ATCC CRL-2638) and 4T1 (Cat. CRL-2539) cell lines were obtained from UACC; 4T1-Luc2 (Cat. CRL-2539-LUC2) cells were purchased from ATCC; SU­DHL­4 (Cat. CRL-2957) cell was provided by Professor Catharine Smith at The University of Arizona; these cell lines were cultured in complete RPMI-1640 medium.

Techniques: Injection, Optical Imaging, Ex Vivo

Journal: Nature Communications

Article Title: Cholesterol-modified sphingomyelin chimeric lipid bilayer for improved therapeutic delivery

doi: 10.1038/s41467-024-46331-7

Figure Lengend Snippet: a A table delineating the physicochemical characterizations of DOX/Lipo with eq. 35 mol % Chol (eq. 64.4 mol % Chol for DOX/Lipo-PChcPC) and 5 mol % DSPE-PEG 2K (n = 3 independent experiments). b – f anti-TNBC effects in metastatic orthotopic 4T1-Luc2 tumor mouse model. A total of 2 × 10 5 cells were injected into the 4 th mammary fat pad of BABL/c mice (n = 5 mice) . On day 15, the mice with primary tumors ~200 mm 3 ( b ) received an i.v. administration of various DOX/Lipo or Doxil at 15 mg DOX/kg. c Average tumor growth curves measured by a digital caliper. d mice BLI on day 15, 20, 29, and 35 by Lago optical imaging. Ex vivo lung metastasis BLI from all 5 mice in each group ( e ) and tumor-bearing mice images ( f ) were taken on day 35. Data in a (right portion), c (n = 5 mice) are expressed as mean ± s.d. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test; survival curves were compared using the log-rank Mantel–Cox test. Source data are provided as a Source Data file.

Article Snippet: CT26 (Cat. ATCC CRL-2638) and 4T1 (Cat. CRL-2539) cell lines were obtained from UACC; 4T1-Luc2 (Cat. CRL-2539-LUC2) cells were purchased from ATCC; SU­DHL­4 (Cat. CRL-2957) cell was provided by Professor Catharine Smith at The University of Arizona; these cell lines were cultured in complete RPMI-1640 medium.

Techniques: Injection, Optical Imaging, Ex Vivo

Journal: Oncoimmunology

Article Title: Combination blockade of KLRG1 and PD-1 promotes immune control of local and disseminated cancers

doi: 10.1080/2162402X.2021.1933808

Figure Lengend Snippet: KLRG1 therapy alone decreases 4T1 tumor burden in the lungs while double blockade therapy decreases subcutaneous 4T1 tumor burden

Article Snippet: 4T1 (purchased from ATCC, Cat# CRL-2539) were cultured in Roswell Park Memorial Institute medium (RPMI-1640) with L-glutamine (2.05 mM/L) (Thermo Fisher Scientific, Cat# SH30027.01).

Techniques: