Immunological and Nonimmunological Effects of Indoleamine 2,3-Dioxygenase on Breast Tumor Growth and Spontaneous Metastasis Formation

The role of the tryptophan-catabolizing enzyme, indoleamine 2,3-dioxygenase (IDO1), in tumor escape and metastasis formation was analyzed using two pairs ofIdo1+andIdo1−murine breast cancer cell lines.Ido1expression in 4T1 cells was knocked down by shRNA, andIdo1expression in NT-5 cells was upregulated by stable transfection. Growth ofIdo1−tumors and spontaneous metastasis formation were inhibited in immunocompetent mice. A higher level of cytotoxic T lymphocytes was generated by spleen cells from mice bearingIdo1−tumors thanIdo1+tumors. Tumor and metastatic growth was enhanced in immunodeficient mice, confirming an intensified immune response in the absence ofIdo1expression. However,Ido1+tumors grow faster thanIdo1−tumors in immunodeficient SCID/beige mice (lacking T, B, and NK cells) suggesting that someIdo1-controlled nonimmunological mechanisms may be involved in tumor cell growth regulation.In vitroexperiments demonstrated that downregulation ofIdo1in tumor cells was associated with decreased cell proliferation, increased apoptosis, and changed expression of cell cycle regulatory genes, whereas upregulation ofIdo1in the cells had the opposite effects. Taken together, our findings indicate thatIdo1expression could exert immunological and nonimmunological effects in murine breast tumor cells.

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Productive Infection of Human Breast Cancer Cell Lines with Human Cytomegalovirus (HCMV)

Pathogens ◽

10.3390/pathogens10060641 ◽

2021 ◽

Vol 10 (6) ◽

pp. 641

Author(s):

Kaitlin M. Branch ◽

Erica C. Garcia ◽

Yin Maggie Chen ◽

Matthew McGregor ◽

Mikayla Min ◽

...

Keyword(s):

Breast Cancer ◽

Tumor Cells ◽

Cell Lines ◽

Cancer Cell ◽

Breast Cancer Cell ◽

Breast Tumor ◽

Cancer Cell Lines ◽

Breast Cancer Cell Lines ◽

Human Breast ◽

Breast Tumor Cells

Breast cancer is the leading cause of cancer deaths among women worldwide. There are many known risk factors for breast cancer, but the role of infectious disease remains unclear. Human cytomegalovirus (HCMV) is a widespread herpesvirus that usually causes little disease. Because HCMV has been detected in breast tumor biopsy samples and is frequently transmitted via human breast milk, we investigated HCMV replication in breast tumor cells. Four human breast cancer cell lines with different expression profiles for the key diagnostic markers of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), were infected with a bacterial artificial chromosome-derived HCMV clinical strain TB40/E tagged with green fluorescent protein (GFP). Fluorescence microscopy confirmed that all four breast cancer cell lines supported virus entry. RNA was isolated from infected cells and the expression of immediate early (UL123), early (UL54), and late (UL111A) genes was confirmed using PCR. Viral proteins were detected by immunoblotting, and viral progeny were produced during the infection of breast tumor cells, as evidenced by subsequent infection of fibroblasts with culture supernatants. These results demonstrate that breast tumor cells support productive HCMV infection and could indicate that HCMV replication may play a role in breast cancer progression.

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Promising potential of [177Lu]Lu-DOTA-folate to enhance tumor response to immunotherapy—a preclinical study using a syngeneic breast cancer model

European Journal of Nuclear Medicine and Molecular Imaging ◽

10.1007/s00259-020-05054-9 ◽

2020 ◽

Author(s):

Patrycja Guzik ◽

Klaudia Siwowska ◽

Hsin-Yu Fang ◽

Susan Cohrs ◽

Peter Bernhardt ◽

...

Keyword(s):

Tumor Growth ◽

Survival Time ◽

Tumor Cells ◽

Median Survival ◽

Breast Tumor ◽

Median Survival Time ◽

Therapy Outcome ◽

Minor Effect

Abstract Purpose It was previously demonstrated that radiation effects can enhance the therapy outcome of immune checkpoint inhibitors. In this study, a syngeneic breast tumor mouse model was used to investigate the effect of [177Lu]Lu-DOTA-folate as an immune stimulus to enhance anti-CTLA-4 immunotherapy. Methods In vitro and in vivo studies were performed to characterize NF9006 breast tumor cells with regard to folate receptor (FR) expression and the possibility of tumor targeting using [177Lu]Lu-DOTA-folate. A preclinical therapy study was performed over 70 days with NF9006 tumor-bearing mice that received vehicle only (group A); [177Lu]Lu-DOTA-folate (5 MBq; 3.5 Gy absorbed tumor dose; group B); anti-CTLA-4 antibody (3 × 200 μg; group C), or both agents (group D). The mice were monitored regarding tumor growth over time and signs indicating adverse events of the treatment. Results [177Lu]Lu-DOTA-folate bound specifically to NF9006 tumor cells and tissue in vitro and accumulated in NF9006 tumors in vivo. The treatment with [177Lu]Lu-DOTA-folate or an anti-CTLA-4 antibody had only a minor effect on NF9006 tumor growth and did not substantially increase the median survival time of mice (23 day and 19 days, respectively) as compared with untreated controls (12 days). [177Lu]Lu-DOTA-folate sensitized, however, the tumors to anti-CTLA-4 immunotherapy, which became obvious by reduced tumor growth and, hence, a significantly improved median survival time of mice (> 70 days). No obvious signs of adverse effects were observed in treated mice as compared with untreated controls. Conclusion Application of [177Lu]Lu-DOTA-folate had a positive effect on the therapy outcome of anti-CTLA-4 immunotherapy. The results of this study may open new perspectives for future clinical translation of folate radioconjugates.

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Breast Tumor Cell Lines From Pleural Effusions2

JNCI Journal of the National Cancer Institute ◽

10.1093/jnci/53.3.661 ◽

1974 ◽

Vol 53 (3) ◽

pp. 661-674 ◽

Cited By ~ 562

Author(s):

R. Cailleau ◽

R. Young ◽

M. Olivé ◽

W. J. Reeves

Keyword(s):

Chromosome Number ◽

Tumor Cell ◽

Tumor Cells ◽

Cell Lines ◽

Breast Tumor ◽

Tumor Cell Lines ◽

Breast Cancer Patients ◽

Pleural Effusions ◽

Epithelial Tumor

Summary During 1973, 4 new epithelial tumor cell lines were isolated from pleural effusions from breast cancer patients. We describe 3 of these lines: MDA-MB-134, with a mean chromosome number of 43; MDA-MB-175, with a mean chromosome number of 49; and MDA-MB-231, with a mean chromosome number between 65 and 69. We isolated the same cell type from 4 of 10 effusions from MDA-MB-134 and from 6 of 8 effusions from MDA-MB-175. We found that pleural effusions as a source of breast tumor cells to be cultured and studied in vitro have the following advantages: 1) large amounts of material and the possibility of obtaining sequential samples from the same patient; 2) high viability of tumor cells; 3) scarcity or absence of fibroblasts; and 4) the possibility of separating the tumor cells from other “contaminating” cell types by differences in their speed or degree of attachment to the flask. All lines from different patients differed, as seen grossly and microscopically. All lines from sequential pleural effusions from the same patient were apparently alike. No viruses or mycoplasmas were detected in any line.

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TGFBI expression reduces in vitro and in vivo metastatic potential of lung and breast tumor cells

Cancer Letters ◽

10.1016/j.canlet.2011.04.010 ◽

2011 ◽

Vol 308 (1) ◽

pp. 23-32 ◽

Cited By ~ 33

Author(s):

Gengyun Wen ◽

Michael A. Partridge ◽

Bingyan Li ◽

Mei Hong ◽

Wupeng Liao ◽

...

Keyword(s):

Tumor Cells ◽

Breast Tumor ◽

Metastatic Potential ◽

Breast Tumor Cells

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EBV-Encoded Mirnas Facilitate Inflammation and Tumor Formation Through Both Intra- and Inter-Cellular Regulation In Vivo Mouse Model

Blood ◽

10.1182/blood.v122.21.3735.3735 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3735-3735

Author(s):

Natsuko Yamakawa ◽

Jun Ogata ◽

Takashi Yahata ◽

Jun Lu ◽

Kazuaki Yokoyama ◽

...

Keyword(s):

Cell Proliferation ◽

Tumor Cells ◽

Small Rnas ◽

Conflicts Of Interest ◽

Functional Characterization ◽

Tumor Formation ◽

Sequencing Analysis ◽

Immunodeficient Mice

Abstract Introduction EB virus (EBV) is associated with heterogeneous lymphomas. Hodgkin's lymphoma (HL) cells are embedded in non-neoplastic bystanders: B, T cells, and macrophages. Without these bystander cells, the lymphoma cells are incapable of being engrafted in immunodeficient mice. In this context, the bystanders are tumor-supportive “inflammatory niche”. Recently, EBV-infected cells produce exosomes that contain EBV specifically encoded miRNAs (EBV-miRNAs). The miRNAs are transferred to cells, and involved in tumor metastasis. However, the detailed mechanism is unknown. Accordingly, we hypothesized that exosomal EBV-miRNAs might redirect tumor surrounding immune cells from tumor reactive into tumor-supportive “inflammatory niche”. Methods We evaluated the expression of EBV-miRNAs in EBV+HL clinical specimens by in situ hybridization, their functional characterization in vitro, and their effects on persistent infection and tumor development in vivo humanized NOG mice model. Moreover, in order to clarify its sorting mechanism, trans factor and cis factor which determined secreted and non-secreted miRNAs was analyzed by use of mass-spectrograhy and next-generation sequencing. Results and Discussion The EBV-miRNAs effects were potent on monocyte/macrophage Mo/Mf in inducing CD69, IL-10, and TNF, suggesting that EBV-miRNAs might polarize Mo/Mf into tumor associated Mf (TAM). EBV-miRNAs suppress tumor cell proliferation in vitro, implying that it works as tumor-suppressor in the tumor cells, while they are required to develop LPD in vivo, which seems contradict to the result in vitro. These results suggest that EBV-miRNAs intra-cellularly regulate the tumor cells to adjust to the surrounding circumstances, for example, to escape from immune surveillance, and inter-cellularly regulate Mo/Mf to support the tumor survival or development. Most importantly, exosomal EBV-miRNAs derived from the tumor cells were transferred to Mf in human EBV+ HL samples. Interestingly, one EBV coded miRNA was not secreted at all, though it abundantly expresses in the cells. The miRNA has been reported to strongly promote cell proliferation in EBV infected tumor cells. It made us hypothesized that the sorting system of secretary and non-secretary miRNAs is critical in the formation of “inflammatory niche”. In order to clarify the mechanism of the sorting, the chimeric miRNA was constructed then, we determined the sequence, which regulates secretion and non-secretion, and purified the protein complex, which specifically bound to the sequence. Mass spectrography and successive knockdown assay, the trans factor which inhibits secretion was identified. Moreover, the next sequencing analysis for the small RNAs revealed that abundant EBV-coded small RNAs occupied RNA-induced silencing complex (RISC), and that non-secreted EBV-miRNA was specifically modified. It is now under investigation whether the modification is involved in the sort mechanism between secretary and non-secretary miRNAs. Taken together, EBV-miRNAs have critical roles in intra- and inter-cellular manner. Especially, the functions as an inter-cellular communicator might be important in the tumor formation and the mechanism needs further investigation. Disclosures: No relevant conflicts of interest to declare.

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Chemoprotection by VEGF: Rationale for combination nab-paclitaxel and bevacizumab

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.1064 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 1064-1064 ◽

Cited By ~ 1

Author(s):

V. Trieu ◽

S. Ran ◽

C. Bivens ◽

N. Desai

Keyword(s):

Protective Effect ◽

Tumor Cells ◽

Breast Tumor ◽

T Test ◽

Receptor Expression ◽

Vegf Receptor ◽

Vegf Expression ◽

Clonogenic Assays

1064 Background: Nanoparticle albumin-bound (nab-) paclitaxel (ABX) has shown greater efficacy and less toxicity than solvent-based paclitaxel (TAX) in xenograft models and clinical trials. The goal of this study was to determine the effects of VEGF modulation in human MDA-MB-231 breast tumor cell line and the effects of ABX and VEGF-neutralizing antibody bevacizumab (AVA) combination on the growth and metastasis of orthotopically implanted MDA-MB-231 tumors. Methods: VEGF expression was evaluated by ELISA in MDA-MB-231 tumor extract one week after treatment (qdx5) with saline, doxorubicin (10 mg/kg), TAX (10 mg/kg), or ABX (15 mg/kg). VEGF-receptor expression in MDA-MB-231 was quantitated by RT-PCR. MDA-MB-231 cytotoxicity with ABX, VEGF, AVA alone or in combination was measured by cytotoxicity and clonogenic assays. Implanted MDA-MB-231 tumors expressing luciferase were treated with saline, 2 cycles of ABX (10 mg/kg, two qdx5 cycles separated by 1 week, N=5) alone or in combination with AVA (2, 4 and 8 mg/kg, 2/wkx6). Tumor lymph node and pulmonary metastasis was determined by measuring luciferase activity. Results: Compared with saline, MDA-MB-231 tumors following chemotherapies exhibited significant tumor shrinkage (p≤0.006, t-test) and VEGF induction (p<0.0001, t-test). MDA-MB-231 was shown to express VEGFR2. Exogenous VEGF had a protective effect on MDA-MB-231 tumor cells by reducing cytotoxicity of ABX in both cytotoxicity and clonogenic assays. Sequestration of VEGF with AVA increased cytotoxicity of ABX in vitro. Treatment of MDA-MB-231 breast tumors with ABX and AVA combination resulted in greater than additive antitumor response and significantly reduced metastasis to the lungs (p=0.025 vs control) and LN (p=0.022) at the highest AVA dose. Conclusions: Chemotherapies induced VEGF expression in MDA-MD-231 breast tumor in vivo. In vitro, VEGF exerted a protective effect against ABX chemotherapy in VEGFR2-expressing MDA-MD-231 cells, which was abrogated by addition of AVA. In vivo, ABX and AVA combination significantly inhibited the metastatic potential of MDA-MB-231 tumor cells. These data provide a rational basis for the combination of nab- paclitaxel and bevacizumab in VEGF-receptor expressing tumors. [Table: see text]

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Hodgkin lymphoma therapy with interleukin-4 receptor–directed cytotoxin in an infiltrating animal model

Blood ◽

10.1182/blood-2004-08-3216 ◽

2005 ◽

Vol 105 (9) ◽

pp. 3707-3713 ◽

Cited By ~ 14

Author(s):

Mariko Kawakami ◽

Koji Kawakami ◽

Mitomu Kioi ◽

Pamela Leland ◽

Raj K. Puri

Keyword(s):

Animal Model ◽

Hodgkin Lymphoma ◽

Cell Lines ◽

Interleukin 4 ◽

Spontaneous Metastasis ◽

Pseudomonas Exotoxin ◽

Immunodeficient Mice ◽

Interleukin 4 Receptor ◽

Lymphoma Therapy

AbstractHodgkin lymphoma represents unique clinicopathologic features because Hodgkin and Reed-Sternberg (H-RS) cells produce a variety of cytokines, express a variety of cytokine receptors, and are surrounded by numerous nonmalignant immunoreactive cells. We found that receptors for interleukin-4 (IL-4R) are highly expressed in H-RS cells. To target interleukin-4 receptor (IL-4R), we used a recombinant protein fusing circularly permuted human IL-4 and Pseudomonas exotoxin termed IL438-37-PE38KDEL, or IL-4 cytotoxin. The cytotoxic effect of IL-4 cytotoxin on H-RS cell lines was determined to be moderate to high in vitro. We developed an infiltrating model of Hodgkin disease (HD) by injecting an adherent population of HD-MyZ cells subcutaneously into the flanks of beige/nude/X-linked immunodeficient mice. The animal model exhibited spontaneous metastasis of H-RS cells to lymph nodes and dissemination to vital organs, including the lungs. Intraperitoneal or intratumoral treatment of these mice with IL-4 cytotoxin resulted in regression of the primary tumor mass and a decrease in the incidence of lymph node metastasis. Mice injected with HD-MyZ cells demonstrated 203% prolonged survival (mean survival, 63 days) compared with control (mean survival, 31 days) when they received systemic IL-4 cytotoxin treatment. Because numerous H-RS cell lines express receptors for IL-4, IL-4 cytotoxin may be a unique agent for the treatment of Hodgkin lymphoma.

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Breast tumor cells isolated from in vitro resistance to trastuzumab remain sensitive to trastuzumab anti-tumor effects in vivo and to ADCC killing

Cancer Immunology Immunotherapy ◽

10.1007/s00262-009-0700-0 ◽

2009 ◽

Vol 58 (11) ◽

pp. 1887-1896 ◽

Cited By ~ 35

Author(s):

Timothy E. Kute ◽

Lori Savage ◽

John R. Stehle ◽

Jung W. Kim-Shapiro ◽

Michael J. Blanks ◽

...

Keyword(s):

Tumor Cells ◽

Breast Tumor ◽

Breast Tumor Cells

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Circulating Tumor Cells Develop Resistance to TRAIL-Induced Apoptosis through Autophagic Removal of Death Receptor 5: Evidence from an In Vitro Model

10.20944/preprints201811.0421.v1 ◽

2018 ◽

Author(s):

Julianne D. Twomey ◽

Baolin Zhang

Keyword(s):

Tumor Cells ◽

Circulating Tumor Cells ◽

Immune Surveillance ◽

Death Receptor ◽

Metastatic Potential ◽

Breast Cancer Cell Lines ◽

Autophagic Flux ◽

Death Receptor 5 ◽

Molecular Features

Circulating tumor cells (CTCs) in the peripheral blood are the precursors to distant metastasis but the underlying mechanisms are poorly understood.  This study aims at understanding the molecular features within CTCs in relation to their metastatic potential.  Using in vitro CTC models, in which breast cancer cell lines are cultured in non-adherent conditions simulating the microenvironment in the blood stream, we found that suspension culture resulted in resistance to TNF-related apoptosis inducing ligand (TRAIL)-mediated cell death. Such a resistance was directly correlated with a reduction in surface and total levels of DR5 protein. In the non-adherent state, cells underwent rapid autophagic flux characterized by an accumulation of autophagosome organelles. Notably, DR5 was translocated to autophagosomes and underwent lysosomal degradation.  Our data suggest that CTCs may evade TNF cytokine mediated immune surveillance through downregulation of DR expression. The data warrants further studies in cancer patients to find the status of DRs and other molecular features within primary CTCs in relation to disease progression or chemoresistance.

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Heat Shock Protein 70 Inhibitor, Alone or in Combination with Bortezomib, Prevented Plasmacytoma Development in Immunodeficient Mice Transplanted with Myeloma Cell Lines

Blood ◽

10.1182/blood.v128.22.5658.5658 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5658-5658

Author(s):

Mariana Bleker de Oliveira ◽

Angela Isabel Eugenio ◽

Veruska Lia Fook Alves ◽

Daniela Zanatta ◽

Mihoko Yamamoto ◽

...

Keyword(s):

Cell Line ◽

Tumor Cells ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Conflicts Of Interest ◽

Control Group ◽

Immunodeficient Mice ◽

Late Apoptosis

Abstract Introduction: HSP70 has an integrative role in protein degradation due to the interaction with many pathways, such as ubiquitin proteasome (UPS), unfolded protein response (UPR) and autophagy. In multiple myeloma (MM) HSP70 is overexpressed and helps to prevent proteotoxic stress and cell death caused by overload of unfolded/misfolded proteins produced by tumor cells. Aims: To explore the role of HSP70 inhibition, isolated or in association with proteasome inhibitor, as therapeutic strategy for MM through in vitro and in vivo analyses. Methods: RPMI8226-LUC-PURO and U266-LUC-PURO bioluminescent cell lines were treated with HSP70 inhibitor (VER155008- 50 μM or 80μM) and proteasome inhibitor (bortezomib 100nM) for evaluation of apoptosis induction by flow cytometry using annexin V and propidium iodide. NOD.Cg-rkdcscid Il2rgtm1Wjl/SzJ immunodeficient mice were used for plasmacytoma xenograft model and treated with intravenous VER155008 (40mg/kg) and bortezomib (1mg/kg), immediately after transplant of RPMI8226-LUC-PURO and U266-LUC-PURO bioluminescent cell lines (N=3 for each group, including controls, bortezomib, VER155008, and combination of bortezomib and VER155008). Bioluminescence was measured in IVIS Kinetic (Capiler Life Science) once a day for seven days. Results: Bortezomib used as single treatment was able to induce apoptosis in RPMI8226-LUC-PURO cell line: the best result for in vitro studies RPMI8226-LUC-PURO was 65% of late apoptosis after treatment with bortezomib. On the other hand, U266-LUC-PURO cell line presented higher percentage of apoptosis when treated with bortezomib and VER155008 combination: U266-LUC-PURO cell line presented more than 60% of late apoptosis after VER155008 (80μM) combined with bortezomib, showing that inhibition of HSP70 could overcome U266-LUC-PURO resistance to bortezomib alone. Mice treated with VER155008, alone or in combination with bortezomib, showed complete inhibition of tumor growth (absence of bioluminescence) for both cell lines when compared with control group after one week of treatment (p<0.001, Two-way ANOVA). Therefore, in vivo studies using mice treated with VER155008, alone or in combination with bortezomib, prevented tumor development after one week of treatment, independent of the cell line used in the xenotransplant. Conclusion: Our study shows that HSP70 and proteasome inhibitors combination induced apoptosis in tumor cells in vivo for both MM cell lines. Since HSP70 is overexpressed in MM and connects several signaling pathways that maintain cell survival, such as UPS, UPR and autophagy, it can represent a key role to establish a new approach for the treatment of MM. Financial support: FAPESP 2010/17668-6 and CNPq (155272/2013-6). UNIFESP Ethics Committee (0219/12). Disclosures No relevant conflicts of interest to declare.

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