Journal: Scientific Reports

Article Title: LOXL2 drives epithelial-mesenchymal transition via activation of IRE1-XBP1 signalling pathway

doi: 10.1038/srep44988

Figure Lengend Snippet: ( a ) Promoter assays were performed in control mouse Eo771 (pLKO) (left) and human MDA-MB-231 cells (shEGFP) (right) and in cells silenced for LOXL2 (shLOXL2). Error bars represent the s.e.m. (n = 3) (*p < 0.05, **p < 0.01, ***p < 0.001). ( b ) LOXL2 knockdown in Eo771 and MDA-MB-231 cells abrogates XBP1 splicing. Cells used in ( a ) were processed for RT-PCR analysis of spliced XBP1. GAPDH levels serve as loading control. Unspliced ( XBP1 u) and spliced ( XBP1s ) forms of XBP1 are indicated. (*) XBP1 hybrid band. One representative RT-PCR analysis of three independent experiments is shown.

Article Snippet: Human HEK293T, MDA-MB-231 and Hs578T, mouse Eo771 and dog MDCK-II cell lines were obtained from the American Type Culture Collection and grown in DMEM media (Gibco), supplemented with 10% foetal bovine serum, 10 mmol/L glutamine (Life Technologies) and 1% penicillin/streptomycin (Invitrogen).

Techniques: Control, Knockdown, Reverse Transcription Polymerase Chain Reaction

Journal: Theranostics

Article Title: Evaluation of cancer immunotherapy using mini-tumor chips

doi: 10.7150/thno.71761

Figure Lengend Snippet: Profiling of tumor responses to anti-PD1 blockade using the mini-tumor chip. (A) Treatment scheme of EO771 tumor-bearing mice. (B) Orthotopic EO771 tumor growth curve with representative responder (RS) and non-responder (NR) tumor. (C) Live/dead staining images of RS and NR tumors with on-chip anti-PD1 treatment for 12 h and quantitative analysis. Scale bar: 500 µm. (D) Viability of RS and NR tumors on-chip. Data points from anti-PD1 treated groups were compared with corresponding controls at the same timepoint by student's t-test (n = 20, **p < 0.01, ****p < 0.001), viability data points of representative images shown in Fig C were highlighted in red color. (E) Flow analysis of CD4+ and CD8+ T cell composition of EO771 dissociated primary tumors and on-chip cell components pre-treatment with both RS and NR tumors.

Article Snippet: To establish PD-L1 overexpression EO771 cell line, EO771 cells were transfected pUNO1-mCD274 vector expressing mouse PD-L1 (Invivogen #puno1-mcd274).

Techniques: Staining

Journal: Theranostics

Article Title: Evaluation of cancer immunotherapy using mini-tumor chips

doi: 10.7150/thno.71761

Figure Lengend Snippet: Prediction of tumors' responses to anti-PD1 treatment using the mini-tumor chip. (A) Experimental scheme test on-chip prediction of primary tumors' responses to anti-PD1 treatment. (B) Dissociated tumor cells on-chip responses to anti-PD1 at day 10, with orthotopic tumors formed by EO771 wild type (WT), PD-L1 overexpression (OE), and PD-L1 knockdown (KD) cells. (C) Tumor growth curve of orthotopic EO771 tumors with wild type, PD-L1 overexpression, and knock-down. Data points from anti-PD1 treated groups were compared with corresponding controls at the same timepoint by student's t-test (n = 20, *p < 0.05, ****p < 0.001).

Article Snippet: To establish PD-L1 overexpression EO771 cell line, EO771 cells were transfected pUNO1-mCD274 vector expressing mouse PD-L1 (Invivogen #puno1-mcd274).

Techniques: Over Expression

Journal: Theranostics

Article Title: Generation of the tumor-suppressive secretome from tumor cells

doi: 10.7150/thno.61006

Figure Lengend Snippet: Generation of iTS cells from osteocytes, osteoclasts, and mammary tumor cells. CM = conditioned medium, CN = control (no CM treatment), β-cat = β-catenin plasmids, A5 = MLO-A5 osteocytes, RAW = RAW 264.7 osteoclasts, EO = EO771 mammary tumor cells, and MDA = MDA-MB-231 breast cancer cells. The single, double, and triple asterisks indicate p < 0.05, p < 0.01, and p < 0.0001, respectively. (A-C) Reduction in EdU-based proliferation and transwell invasion of EO771 mammary tumor cells by A5 osteocyte-derived, RAW264.7 osteoclast-derived, and EO771 breast cancer-derived iTS CMs in 2 days. These cells were transfected with β-catenin plasmids. (D) Inhibition of EdU-based proliferation, scratch-based migration, and transwell invasion of MDA-MB-231 breast cancer cells by β-catenin-overexpressing MDA-MB-231-derived CM in 2 days.

Article Snippet: EO771 mouse mammary tumor cells (CH3 BioSystems, Amherst, NY, USA), 4T1.2 mouse mammary tumor cells (obtained from Dr. R. Anderson at Peter MacCallum Cancer Institute, Melbourne, Australia), PANC-1 human pancreatic cancer cells (ATCC), and MCF-7 human estrogen receptor (ER)-positive breast cancer cells (ATCC) were cultured in DMEM (MT10013CV, Thermo Fisher Scientific).

Techniques: Control, Derivative Assay, Transfection, Inhibition, Migration

Journal: Theranostics

Article Title: Generation of the tumor-suppressive secretome from tumor cells

doi: 10.7150/thno.61006

Figure Lengend Snippet: Inhibition of the growth of cancer tissue fragments and the tumor invasion to the lung by tissue-derived iTS CM. CM = conditioned medium, pl = placebo, β-cat = β-catenin plasmids, and EO = EO771 mammary tumor cells. The double and triple asterisk indicates p < 0.01 and 0.0001, respectively. (A) Shrinkage of breast cancer tissue fragments by β-catenin-overexpressing and BML284-treated MCF7-derived iTS CM in 72 h. (B) Shrinkage of prostate cancer tissue fragments by β-catenin-overexpressing and BML284-treated PC3-derived iTS CM in 72 h. (C) Shrinkage of EO771 mammary tumor spheroid (green) by co-culturing with β-catenin-overexpressing EO771 spheroid (red) and by β-catenin-overexpressing EO771-derived iTS CM in 72 h. (D) Extravasation assay for examining the invasion of tumor cells into the lung. Increase in the invaded cells by inoculating β-catenin-overexpressing EO771 cells, and a decrease by the administration of β-catenin-overexpressing EO771 cell-derived CM. The green fluorescently labeled EO771 cells were injected into the tail vein and mice were sacrificed in 48 h for histological inspection. The black arrows indicate tumor cells.

Article Snippet: EO771 mouse mammary tumor cells (CH3 BioSystems, Amherst, NY, USA), 4T1.2 mouse mammary tumor cells (obtained from Dr. R. Anderson at Peter MacCallum Cancer Institute, Melbourne, Australia), PANC-1 human pancreatic cancer cells (ATCC), and MCF-7 human estrogen receptor (ER)-positive breast cancer cells (ATCC) were cultured in DMEM (MT10013CV, Thermo Fisher Scientific).

Techniques: Inhibition, Derivative Assay, Labeling, Injection

Journal: Theranostics

Article Title: Generation of the tumor-suppressive secretome from tumor cells

doi: 10.7150/thno.61006

Figure Lengend Snippet: Inhibition of tumor growth and bone loss by iTS CM in the mouse model. CM = conditioned medium, pl = placebo, β-cat = β-catenin plasmids, and BML = BML284. The single, double, and triple asterisks indicate p < 0.05, p < 0.01, and p < 0.0001, respectively. (A) Procedure for the inoculation of EO771 cells to the mammary fat pad and tibia. (B) Increase in tumor weight by inoculating β-catenin-overexpressing EO771 cells, and a decrease by the administration of β-catenin-overexpressing EO771 cell-derived CM in 2 weeks (N = 10). (C) Increase in tumor weight by the systemic administration of BML284, and a decrease by the administration of BME284-treated EO771 cell-derived CM in 2 weeks (N = 10). (D) μCT images of the proximal tibia and BV/TV (bone volume ratio), BMD (bone mineral density), Tb.N (trabecular number), and Tb.Sp (trabecular separation) for 5 groups of C57BL/6 female mice (N = 10). They are the placebo (no treatment), inoculation of β-catenin-overexpressing cells (β-cat cells), administration of β-catenin-overexpressing EO771 cell-derived CM (β-catenin CM), inoculation of BML284-treated cells (BML), and administration of BML284-treated cell-derived CM (BML CM).

Article Snippet: EO771 mouse mammary tumor cells (CH3 BioSystems, Amherst, NY, USA), 4T1.2 mouse mammary tumor cells (obtained from Dr. R. Anderson at Peter MacCallum Cancer Institute, Melbourne, Australia), PANC-1 human pancreatic cancer cells (ATCC), and MCF-7 human estrogen receptor (ER)-positive breast cancer cells (ATCC) were cultured in DMEM (MT10013CV, Thermo Fisher Scientific).

Techniques: Inhibition, Derivative Assay

Journal: Theranostics

Article Title: Generation of the tumor-suppressive secretome from tumor cells

doi: 10.7150/thno.61006

Figure Lengend Snippet: Mass spectrometry-based prediction of tumor suppressors and the effect of enolase 1 and ubiquitin C. EO = EO771 mammary tumor cells, CM = conditioned medium, CN = control (no CM treatment), β-cat = β-catenin plasmids, BML = BML284, Eno1 = Enolase 1, and Ubc = ubiquitin C. The single and double asterisks indicate p < 0.05 and p < 0.01, respectively. (A) List of 25 top tumor suppressor candidates identified by mass spectrometry-based proteomics analysis. (B) Reduction in MTT-based proliferation of EO771 mammary tumor cells by 9 recombinant proteins (5 μg/mL) in 48 h. (C) Levels of enolase 1 and ubiquitin C in β-catenin-overexpressing and BML284-treated EO771 CMs by ELISA. (D) Inhibition in the scratch-based migration of EO771 mammary tumor cells by enolase 1 and ubiquitin C in 24 h. (E) Expression of β-catenin and enolase 1 in EO771 cells that were treated with AP-Ⅲ-a4, an inhibitor of enolase 1. (F&G) Repressive effects of AP-Ⅲ-a4 on the proliferation (in 48 h) and migration of EO771 cells (in 24 h) by β-catenin overexpressing iTS CM.

Article Snippet: EO771 mouse mammary tumor cells (CH3 BioSystems, Amherst, NY, USA), 4T1.2 mouse mammary tumor cells (obtained from Dr. R. Anderson at Peter MacCallum Cancer Institute, Melbourne, Australia), PANC-1 human pancreatic cancer cells (ATCC), and MCF-7 human estrogen receptor (ER)-positive breast cancer cells (ATCC) were cultured in DMEM (MT10013CV, Thermo Fisher Scientific).

Techniques: Mass Spectrometry, Ubiquitin Proteomics, Control, Recombinant, Enzyme-linked Immunosorbent Assay, Inhibition, Migration, Expressing

Journal: Theranostics

Article Title: Generation of the tumor-suppressive secretome from tumor cells

doi: 10.7150/thno.61006

Figure Lengend Snippet: Effect of silencing enolase 1 and ubiquitin C. CM = conditioned medium, CN = control (no CM treatment), β-cat = β-catenin plasmids, siEno1 = Enolase 1 siRNA, siUbc = ubiquitin C siRNA, EO = EO771 mammary tumor cells, TR = TRAMP prostate cancer cells, and PA = PANC-1 pancreas cancer cells. The double asterisk indicates p < 0.01. (A) siRNA-mediated knockdown of enolase 1, and ubiquitin C in EO771 breast cancer cells. (B&C) Promotion of MTT-based proliferation, and scratch-based migration of EO771 breast cancer cells by enolase 1 and ubiquitin C siRNA-treated CMs in 2 days. (D-I) Effects of enolase 1 and ubiquitin C siRNAs. Silencing these two proteins significantly prevented the reduction in EdU-based proliferation and Transwell invasion of EO771, TRAMP, and PANC-1 cells by their own β-catenin-overexpressing iTS CMs in 2 days.

Article Snippet: EO771 mouse mammary tumor cells (CH3 BioSystems, Amherst, NY, USA), 4T1.2 mouse mammary tumor cells (obtained from Dr. R. Anderson at Peter MacCallum Cancer Institute, Melbourne, Australia), PANC-1 human pancreatic cancer cells (ATCC), and MCF-7 human estrogen receptor (ER)-positive breast cancer cells (ATCC) were cultured in DMEM (MT10013CV, Thermo Fisher Scientific).

Techniques: Ubiquitin Proteomics, Control, Knockdown, Migration

Journal: Theranostics

Article Title: Generation of the tumor-suppressive secretome from tumor cells

doi: 10.7150/thno.61006

Figure Lengend Snippet: Tumor selectivity and the involvement of CD44. A5 = MLO-A5 osteocytes, β-cat = β-catenin plasmids, BML = BML284, Eno1 = Enolase 1, Ubc = ubiquitin C. The signal and double asterisk indicates p < 0.01 and p < 0.05, respectively. (A) Comparison of MTT-based viability of four non-tumor cells (KTB34, KTB6, MC3T3, MSC) and four tumor cells (EO771, TRAMP, 4T1.2, PANC-1) in response to β-catenin-iTS CM and BML-iTS CM in 2 days. (B) Tumor selectivity of EO771 CM, 4T1.2 CM, TRAMP CM, and PANC-1 CM. MTT-based tumor selectivity is defined as the ratio of (reduction in tumor cells) to (reduction in non-tumor cells). The selectivity value above 1 indicates that MTT-based inhibition is more selective to tumor cells than non-tumor cells. Of note, N.D. = not defined since the viability of non-tumor cells is stimulated. (C&D) Tumor selectivity of Enolase 1 and ubiquitin C. (E) CD44 was co-immunoprecipitated with Eno1. The protein extracts of EO771 cells were incubated with anti-Eno1 antibody using the protein A/G beads. Immunoprecipitates and total cell lysates were analyzed by Western blotting with anti-CD44 and anti-Eno1 antibodies as indicated. (F&G) siRNA knockdown of CD44 suppressed Eno1-mediated inhibition of the proliferation of EO771 cells. (H) siRNA knockdown of CD44 suppressed Eno1-mediated downregulation of MMP9, Runx2, and Snail in EO771 cells.

Article Snippet: EO771 mouse mammary tumor cells (CH3 BioSystems, Amherst, NY, USA), 4T1.2 mouse mammary tumor cells (obtained from Dr. R. Anderson at Peter MacCallum Cancer Institute, Melbourne, Australia), PANC-1 human pancreatic cancer cells (ATCC), and MCF-7 human estrogen receptor (ER)-positive breast cancer cells (ATCC) were cultured in DMEM (MT10013CV, Thermo Fisher Scientific).

Techniques: Ubiquitin Proteomics, Comparison, Inhibition, Immunoprecipitation, Incubation, Western Blot, Knockdown

Journal: Theranostics

Article Title: Generation of the tumor-suppressive secretome from tumor cells

doi: 10.7150/thno.61006

Figure Lengend Snippet: Effects of enolase 1, ubiquitin C, and iTS CM on the expression of tumor-promoting and tumor-suppressing genes. CM = conditioned medium, CN = control (no CM treatment), β-cat = β-catenin plasmids, siEno1 = Enolase 1 siRNA, siUbc = ubiquitin C siRNA, EO = EO771 mammary tumor cells. (A&B) Expression of MMP9, Runx2, Snail, p53, and TRAIL in response to enolase 1 and ubiquitin C in EO771 breast cancer cells. (C&D) Expression of MMP9, Runx2, Snail, p53, and TRAIL in response to β-catenin-overexpressing iTS CM impaired by siRNAs specific to enolase 1 and ubiquitin C. (E) Expression of PDL1 in EO771 mammary tumor cells in response to β-catenin-overexpressing iTS CM, enolase 1, and ubiquitin C. (F&G) Expression of MMP9, Runx2, Snail, p53, TRAIL, and caspase 3 in EO771 mammary tumor cells in response to β-catenin-overexpressing pre-treatment tumor cell-derived CM. (H) Low survival for cancer patients with a high transcript level of MMP9, Runx2, or Snail. (I) Proposed regulatory mechanism to inhibit tumor progression by iTS-CM. According to the mechanism, β-catenin-overexpressing iTS cells secrete ubiquitin C (Ubc), enolase 1 (Eno1), p53, and Trail. They suppress the progression of tumor cells by downregulating MMP9, Runx2, Snail, and PDL1, while upregulating cleaved-caspase 3. It should be noted that Eno1 interacts with CD44 and inhibits MMP9, Runx2, and Snail.

Article Snippet: EO771 mouse mammary tumor cells (CH3 BioSystems, Amherst, NY, USA), 4T1.2 mouse mammary tumor cells (obtained from Dr. R. Anderson at Peter MacCallum Cancer Institute, Melbourne, Australia), PANC-1 human pancreatic cancer cells (ATCC), and MCF-7 human estrogen receptor (ER)-positive breast cancer cells (ATCC) were cultured in DMEM (MT10013CV, Thermo Fisher Scientific).

Techniques: Ubiquitin Proteomics, Expressing, Control, Derivative Assay

Journal: bioRxiv

Article Title: Engineered bacteria recruit and orchestrate anti-tumor immunity

doi: 10.1101/2022.06.16.496462

Figure Lengend Snippet: (A) Tumor growth curves from C57BL/6 mice ( n = 5 per group) subcutaneously implanted with 5 x 10 5 MC38 colorectal tumor cells on both hind flanks. When tumor volumes were 50–150 mm 3 , mice received intratumoral injections (indicated by black arrows) every 3–4 days with PBS, eSLC, or eSLC-hCXCL16 K42A . Data are representative of 4 independent experiments (**** P <0.0001, 2-way ANOVA with Holm-Sidak post-hoc test at final measurement time point). (B–D) Flow cytometric analysis of tumor infiltrating lymphocytes isolated from subcutaneously growing MC38 tumors ( n ≥ 4 mice per group) on (B–C) day 5 or (D) day 8 following intratumoral injections (performed as in (A)) with PBS, eSLC, or eSLC-hCXCL16 K42A . (B) Frequencies of isolated Ki-67 + CD4 + Foxp3” and CD8 + T cells. (C–D) Frequencies of isolated Granzyme-B + CD4 + Foxp3” and Granzyme-B + CD8 + T cells on (C) day 5 post-injection and (D) day 8 post-injection. For (B–D), data are representative of 3 independent experiments (* P <0.05, ** P <0.01, 2-way ANOVA with Holm-Sidak post-hoc test). (E) Tumor growth curves from C57BL/6 mice ( n = 5 per group) subcutaneously implanted with 1 x 10 6 EO771 triple negative breast cancer tumor cells on both hind flanks. When tumor volumes were 50–150 mm 3 , mice received intratumoral injections (indicated by black arrows) every 3–4 days with PBS, eSLC, or eSLC-hCXCL16 K42A . Data are representative of 2 independent experiments (*** P <0.001, 2-way ANOVA with Holm-Sidak post-hoc test at final measurement time point). (F) C57BL/6 mice ( n ≥ 5 per group) subcutaneously implanted with 5 x 10 5 MC38 colorectal tumor cells on both hind flanks. When tumor volume reached 50–150 mm 3 , mice received a single intravenous injection (black arrow) of PBS, eSLIC, or eSLIC-hCXCL16 K42A . Schematic for intravenous treatment experiment (left) and tumor growth curves (right). Data are representative of 2 independent experiments (*** P <0.001, 2-way ANOVA with Holm-Sidak post-hoc test at final measurement time point). All data displayed as mean ± s.e.m.

Article Snippet: Female 7-8 week old BALB/c (for A20 tumors) or C57BL/6N (EO771 and MC38 tumors) mice were purchased from Taconic Biosciences or Jackson Laboratories, allowed to acclimate for a week and then injected with tumor cells.

Techniques: Isolation, Injection

Journal: bioRxiv

Article Title: Engineered bacteria recruit and orchestrate anti-tumor immunity

doi: 10.1101/2022.06.16.496462

Figure Lengend Snippet: (A-B) Individual tumor growth curves from C57BL/6 mice ( n = 5 per group) subcutaneously implanted with (A) 5 x 10 5 MC38 colorectal tumor cells or (B) 1 x 10 6 EO771 triple negative breast cancer cells on both hind flanks. When tumor volumes were 50–150 mm 3 , mice received intratumoral injections (indicated by black arrows) every 3–4 days with PBS, eSLC, or eSLC- hCXCL16 K42A . (A): Data are representative of 4 independent experiments. (B) Data are representative of 2 independent experiments. (C) C57BL/6 mice ( n ≥ 5 per group) subcutaneously implanted with 5 x 10 5 MC38 colorectal tumor cells on both hind flanks. When tumor volume reached 50–150 mm 3 , mice received a single intravenous injection (black arrow) of PBS, eSLIC, or eSLIC-hCXCL16 K42A . Individual tumor growth curves displayed. Data are representative of 2 independent experiments.

Article Snippet: Female 7-8 week old BALB/c (for A20 tumors) or C57BL/6N (EO771 and MC38 tumors) mice were purchased from Taconic Biosciences or Jackson Laboratories, allowed to acclimate for a week and then injected with tumor cells.

Techniques: Injection

Journal: bioRxiv

Article Title: Engineered bacteria recruit and orchestrate anti-tumor immunity

doi: 10.1101/2022.06.16.496462

Figure Lengend Snippet: (A) Schematic overview of approach to recruit dendritic cells to tumors via CCL20 production in probiotic bacteria to complement activated T cell recruitment via CXCL16 (as in ). (B) Tumor growth curves from C57BL/6 mice ( n = 5 per group) subcutaneously implanted with 5 x 10 5 MC38 colorectal tumor cells on both hind flanks. When tumor volumes were 50–150 mm 3 , mice received intratumoral injections (indicated by black arrows) every 3–4 days with PBS, eSLC, eSLC-hCXCL16 K42A , eSLC-CCL20, or eSLC-combo (1:1 mixture of eSLC-hCXCL16 K42A and eSLC-CCL20). Data are representative of 4 independent experiments (*** P <0.001, **** P <0.0001, 2-way ANOVA with Holm-Sidak post-hoc test at final measurement time point). (C–E) Flow cytometric analysis of tumor infiltrating immune cells isolated from subcutaneously growing MC38 tumors ( n ≥ 4 mice per group) following intratumoral injections (performed as in (B)) with PBS, eSLC, eSLC-hCXCL16 K42A , or eSLC-combo (as in (A)). (C) Frequencies and (D) numbers of type 1 conventional dendritic cells on day 5 post-treatment. (E) Frequencies of intratumoral Granzyme-B + CD8 + T cells. Data are representative of 3 independent experiments (* P <0.05, 1-way ANOVA with Holm-Sidak post-hoc test). All data displayed as mean ± s.e.m.

Article Snippet: Female 7-8 week old BALB/c (for A20 tumors) or C57BL/6N (EO771 and MC38 tumors) mice were purchased from Taconic Biosciences or Jackson Laboratories, allowed to acclimate for a week and then injected with tumor cells.

Techniques: Isolation

Journal: bioRxiv

Article Title: Engineered bacteria recruit and orchestrate anti-tumor immunity

doi: 10.1101/2022.06.16.496462

Figure Lengend Snippet: Individual tumor growth curves from C57BL/6 mice ( n = 5 per group) subcutaneously implanted with 5 x 10 5 MC38 colorectal tumor cells on both hind flanks. When tumor volumes were 50–150 mm 3 , mice received intratumoral injections (indicated by black arrows) every 3–4 days with PBS, eSLC, eSLC-hCXCL16 K42A , eSLC-CCL20, or eSLC-combo (1:1 mixture of eSLC-hCXCL16 K42A and eSLC-CCL20). Data are representative of 4 independent experiments.

Article Snippet: Female 7-8 week old BALB/c (for A20 tumors) or C57BL/6N (EO771 and MC38 tumors) mice were purchased from Taconic Biosciences or Jackson Laboratories, allowed to acclimate for a week and then injected with tumor cells.

Techniques: