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Aberrant glycolysis associates with inflammatory tumour microenvironment and promotes metastasis in triple negative breast cancer
abstracts
Annals of Oncology zones of spheroids from 12 to 60%. 2 min. of US exposure decreased the amount of DOX in these zones. US also increased DOX, 5-FU and PTX toxicity in cancer cell spheroids 8-, 1.2- and 1.5-fold, respectively. Conclusions: US is a promising physical method to enhance anticancer drug efficacy, especially into 3D cell cultures. However, there is a lack of evidence about its efficacy and further studies are needed. Legal entity responsible for the study: The authors. Funding: Lithuanian University of Health Sciences; Kaunas University of Technology. Disclosure: All authors have declared no conflicts of interest. Tr1-like cells in human peripheral blood are part of the T effector memory pool and are preferentially stimulated via CD55
I.A. Charles, J.M. Ramage, I. Spendlove Academic Oncology, City Hospital Campus - Nottingham University Hospitals NHS Trust, Nottingham, UK Background: Type 1 regulatory (Tr1) T cells, like every effector T cells, arise from stimulation of naı¨ve T cell precursor and enter a long term memory pool. While they can adapt their function to specific environmental cues, sometimes called plasticity, their phenotype remains broadly fixed. Unlike natural T regulator cells that emerge from the thymus with a defined phenotype (CD4þCD25hiCD127loFoxP3þ), there is uncertainty over the characterisation of inducible Tr1 cells. Methods: In this study we stimulated human total CD4þ T cells with PMA/ Ionomycin and also demonstrated that these cells respond specifically to costimulation via CD97CD55. Dual cell surface capture (CSA) was used to assay for IL-10 and IFN-c and intracellular staining for Tr1 markers. Results: A small (<5%) population were IL-10þ IL-4-, IFN-c- and expressed other markers commonly associated with Tr1-like cells. These included; CD49b, LAG-3, CD226, PD-1, CTLA-4, and TIM-3. However they were negative for FoxP3 expression, and expressed Cmaf, which is thought to be responsible for the transcriptional events within this population. Furthermore, we show that these Tr1-like cells form part of the immunological memory and reside predominantly within the effector memory (CD62L- CD45ROþ, TEM) pool. Unlike the majority of other studies where Tr1 is generated by chronic stimulation of PBMCs in the presence of recombinant IL-10 for several days, as far as we are aware, this is the first report characterising Tr1 directly ex vivo from human peripheral blood. Conclusions: We have also demonstrated that these cells respond specifically to costimulation via CD97-CD55 to drive proliferation and maintain the IL-10 single positive, Tr1 phenotype outlined above. This supports the idea that once differentiated from naı¨ve precursors, Tr1-like cells respond to CD55 costimulation to maintain a small (<5%) pool with a committed IL-10þ IL-4-IFN-c- phenotype. Legal entity responsible for the study: The University of Nottingham. Funding: The Commonwealth Scholarships Commission, London. Disclosure: All authors have declared no conflicts of interest.
16P
Functional analysis of tumour infiltrating lymphocytes in triple negative breast cancer focusing on granzyme B
H. Kawaji1, M. Kubo2, Y. Motoyama1, A. Shimazaki1, S. Hayashi1, K. Kurata1, M. Yamada2, K. Kaneshiro1, M. Kai2, M. Nakamura2 1 Surgery and Oncology, Kyushu University - Graduate School of Medical Sciences Faculty of Medical Sciences, Fukuoka, Japan, 2Surgery And Oncology, Kyushu University Hospital, Fukuoka, Japan Background: Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes in breast cancer and has a worse prognosis than others. Tumor infiltrating lymphocytes (TIL) are considered to be a prognostic factor in TNBC. Cytotoxic T cells (CTL) produce cytokines and cytotoxic substances such as perforin and granzyme B, and attack target cells. We have previously shown that combination of PD-L1 expression and TIL is useful as a functional indicator of tumor immune activation. Granzyme B released from CTL induces apoptosis by passing through pores formed by perforin on the cell membrane of target cells. We focused on the cytotoxic substance granzyme B and explored functional of TIL. Methods: This study included 228 patients with primary TNBC who underwent resection without neoadjuvant chemotherapy at Kyushu University Hospital (Japan), between January 2004 and December 2014. We retrospectively analyzed granzyme B, CD8, PD-L1 expression assessed by immunohistochemistry and TIL in 228 TNBCs. Granzyme B was evaluated in TIL, 3% was defined as high expression, and < 3% as low. We also explored the correlation between immunologic features on tumors and immune cells and the clinicopathological characteristics of the tumors, their response to chemotherapy and clinical outcome. Results: Among the 228 tumors, granzyme B expression was classified as high in 106 (46.5%), low in 122 (53.5%). Granzyme B-high is correlated with high levels of TIL (p ¼ 0.004), CD8 expression of T cell (p ¼ 0.016) and PD-L1 of tumor cells (p<.0001). In the TIL-high and granzyme B-high group, it is considered that the anti-tumor immune system is activated, and it tend to be the most favorable prognosis. On the other hand, in the TIL-high and granzyme B-low group, despite lymphocyte migration,
Volume 30 | Supplement 5 | October 2019
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Aberrant glycolysis associates with inflammatory tumour microenvironment and promotes metastasis in triple negative breast cancer
C. Lin Department of Biochemistry and Molecular Cell Biology, Taipei Medical University, Taipei, Taiwan Background: Triple-negative breast cancer (TNBC) is account for 1025% of breast cancer incidence with more aggressive phenotype, metastatic capability, and poorer prognosis than other subtypes. Altered cancer metabolism is an emerging hallmark of cancer. Previous studies reported that TNBC cells exhibit greater aerobic glycolysis than non-TNBC cells; however, the detailed regulatory mechanism is largely unknown. Methods: The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were used to analyze expression of glycolytic genes and clinical relevance in TNBC patients. Immunohistochemistry assay was performed to confirm the expression of glycolytic gene in breast cancer tissues. Gain-of-function and loss-of-function studies were conducted in TNBC cell lines. The effect of inflammatory microenvironment was carried out by coculture of macrophages and TNBC cells, and the expression of inflammatory cytokines were performed by real-time PCR. Results: We compared the differential genes expression in TNBC and non-TNBC by in silico analysis. Interestingly, the expression of glucose transporter 3 (Glut3) is upregulated in TNBC patients, compared to non-TNBC patients. Mechanistically, overexpression of Glut3 regulated the expression of epithelial-mesenchymal transition (EMT) genes and promoted invasiveness and metastasis of TNBC cells. Activation of IL6/ STAT3 signaling regulates the expression of Glut3 and glycolytic genes, and coexpression of IL6/Glut3 rendered poorer survival outcome, particularly in TNBC. Moreover, conditioned medium (CM) from Glut3-expressing tumor cells induced macrophage activation and production of pro-inflammatory cytokines, and patients with high Glut3 level associated with inflammaotry signautres. Conclusions: Upregulation of IL6/STAT3 signaling axis promotes expression of Glut3 and glycolytic genes. Elevation of Glut3 in TNBC induces macrophage activation and production of pro-inflammatory cytokines. Our data show the possible association between glucose metabolism and inflammatory microenvironment in TNBC. Legal entity responsible for the study: The author. Funding: Ministry of Science and Technology, Taiwan. Disclosure: The author has declared no conflicts of interest.
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Anti-cancer effects of differentiation-inducing factor-1 in triple negative breast cancer
F. Tetsuo1, M. Arioka2, F.Y. Takahashi3, F. Nishimura1, T. Sasaguri2 Dental, Kyushu University, Fukuoka, Japan, 2Clinical Pharmacology, Kyushu University, Fukuoka, Japan, 3Pharmacology, University of Occupational and Environmental Health, Kitakyushu, Japan
1
Background: Breast cancer is the most common cancer in women and its metastasis markedly exacerbates patients’ prognosis. In particular, triple negative breast cancer (TNBC), which lacks estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor 2 (HER2), is an aggressive subtype of breast cancer frequently forming metastatic lesions. Differentiation-inducing factor-1 (DIF-1) identified in Dictyostelium discoideum inhibits cell proliferation and migration of various mammalian cancer cells. However, the effect of DIF-1 on TNBC has not been examined. Here, we investigated whether DIF-1 shows anti-proliferative and anti-metastatic effects on TNBC by in-vivo and in-vitro experiments. Methods: In in-vivo experiments, we used cancer xenograft model mice in which murine TNBC 4T1/Luc cells (1.0 106 cells/mL) were injected into mammary fat pads to evaluate the effects of intragastric administration of DIF-1 (300 mg/kg/day) on the primary tumor growth and lung metastasis. In in-vitro experiments, we carried out assays for cell proliferation, migration and invasion to evaluate the anti-proliferative and antimetastatic effects of DIF-1. We also conducted Western blotting and real-time RT-PCR to identify the mechanisms for DIF-1’s anti-cancer effects. Results: In vivo, administration of DIF-1 significantly suppressed the primary tumor growth and lung metastasis without adverse effects such as weight loss and myelosuppression. In vitro, DIF-1 reduced cyclin D1 and c-Myc by inhibiting transcription and promoting degradation of the proteins, which resulted in the suppression of cell proliferation. DIF-1 also suppressed cell migration and invasion and reduced the protein
doi:10.1093/annonc/mdz238 | v5
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz238.016/5576344/ by guest on 07 October 2019
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it is presumed that they do not recognize the antigen, that is, they are in a state of immune tolerance and thus have a poor prognosis. In the group with granzyme B-high, the patients treated with anthracycline as adjuvant chemotherapy significantly improved their prognosis (p ¼ 0.043). The high expression of granzyme B may be a predictor of postoperative chemotherapy. Conclusions: Granzyme B is an important substance of cytotoxic pathway and has been possible to be an indicator of TIL activation. Legal entity responsible for the study: Masafumi Nakamura. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.
Annals of Oncology zones of spheroids from 12 to 60%. 2 min. of US exposure decreased the amount of DOX in these zones. US also increased DOX, 5-FU and PTX toxicity in cancer cell spheroids 8-, 1.2- and 1.5-fold, respectively. Conclusions: US is a promising physical method to enhance anticancer drug efficacy, especially into 3D cell cultures. However, there is a lack of evidence about its efficacy and further studies are needed. Legal entity responsible for the study: The authors. Funding: Lithuanian University of Health Sciences; Kaunas University of Technology. Disclosure: All authors have declared no conflicts of interest. Tr1-like cells in human peripheral blood are part of the T effector memory pool and are preferentially stimulated via CD55
I.A. Charles, J.M. Ramage, I. Spendlove Academic Oncology, City Hospital Campus - Nottingham University Hospitals NHS Trust, Nottingham, UK Background: Type 1 regulatory (Tr1) T cells, like every effector T cells, arise from stimulation of naı¨ve T cell precursor and enter a long term memory pool. While they can adapt their function to specific environmental cues, sometimes called plasticity, their phenotype remains broadly fixed. Unlike natural T regulator cells that emerge from the thymus with a defined phenotype (CD4þCD25hiCD127loFoxP3þ), there is uncertainty over the characterisation of inducible Tr1 cells. Methods: In this study we stimulated human total CD4þ T cells with PMA/ Ionomycin and also demonstrated that these cells respond specifically to costimulation via CD97CD55. Dual cell surface capture (CSA) was used to assay for IL-10 and IFN-c and intracellular staining for Tr1 markers. Results: A small (<5%) population were IL-10þ IL-4-, IFN-c- and expressed other markers commonly associated with Tr1-like cells. These included; CD49b, LAG-3, CD226, PD-1, CTLA-4, and TIM-3. However they were negative for FoxP3 expression, and expressed Cmaf, which is thought to be responsible for the transcriptional events within this population. Furthermore, we show that these Tr1-like cells form part of the immunological memory and reside predominantly within the effector memory (CD62L- CD45ROþ, TEM) pool. Unlike the majority of other studies where Tr1 is generated by chronic stimulation of PBMCs in the presence of recombinant IL-10 for several days, as far as we are aware, this is the first report characterising Tr1 directly ex vivo from human peripheral blood. Conclusions: We have also demonstrated that these cells respond specifically to costimulation via CD97-CD55 to drive proliferation and maintain the IL-10 single positive, Tr1 phenotype outlined above. This supports the idea that once differentiated from naı¨ve precursors, Tr1-like cells respond to CD55 costimulation to maintain a small (<5%) pool with a committed IL-10þ IL-4-IFN-c- phenotype. Legal entity responsible for the study: The University of Nottingham. Funding: The Commonwealth Scholarships Commission, London. Disclosure: All authors have declared no conflicts of interest.
16P
Functional analysis of tumour infiltrating lymphocytes in triple negative breast cancer focusing on granzyme B
H. Kawaji1, M. Kubo2, Y. Motoyama1, A. Shimazaki1, S. Hayashi1, K. Kurata1, M. Yamada2, K. Kaneshiro1, M. Kai2, M. Nakamura2 1 Surgery and Oncology, Kyushu University - Graduate School of Medical Sciences Faculty of Medical Sciences, Fukuoka, Japan, 2Surgery And Oncology, Kyushu University Hospital, Fukuoka, Japan Background: Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes in breast cancer and has a worse prognosis than others. Tumor infiltrating lymphocytes (TIL) are considered to be a prognostic factor in TNBC. Cytotoxic T cells (CTL) produce cytokines and cytotoxic substances such as perforin and granzyme B, and attack target cells. We have previously shown that combination of PD-L1 expression and TIL is useful as a functional indicator of tumor immune activation. Granzyme B released from CTL induces apoptosis by passing through pores formed by perforin on the cell membrane of target cells. We focused on the cytotoxic substance granzyme B and explored functional of TIL. Methods: This study included 228 patients with primary TNBC who underwent resection without neoadjuvant chemotherapy at Kyushu University Hospital (Japan), between January 2004 and December 2014. We retrospectively analyzed granzyme B, CD8, PD-L1 expression assessed by immunohistochemistry and TIL in 228 TNBCs. Granzyme B was evaluated in TIL, 3% was defined as high expression, and < 3% as low. We also explored the correlation between immunologic features on tumors and immune cells and the clinicopathological characteristics of the tumors, their response to chemotherapy and clinical outcome. Results: Among the 228 tumors, granzyme B expression was classified as high in 106 (46.5%), low in 122 (53.5%). Granzyme B-high is correlated with high levels of TIL (p ¼ 0.004), CD8 expression of T cell (p ¼ 0.016) and PD-L1 of tumor cells (p<.0001). In the TIL-high and granzyme B-high group, it is considered that the anti-tumor immune system is activated, and it tend to be the most favorable prognosis. On the other hand, in the TIL-high and granzyme B-low group, despite lymphocyte migration,
Volume 30 | Supplement 5 | October 2019
17P
Aberrant glycolysis associates with inflammatory tumour microenvironment and promotes metastasis in triple negative breast cancer
C. Lin Department of Biochemistry and Molecular Cell Biology, Taipei Medical University, Taipei, Taiwan Background: Triple-negative breast cancer (TNBC) is account for 1025% of breast cancer incidence with more aggressive phenotype, metastatic capability, and poorer prognosis than other subtypes. Altered cancer metabolism is an emerging hallmark of cancer. Previous studies reported that TNBC cells exhibit greater aerobic glycolysis than non-TNBC cells; however, the detailed regulatory mechanism is largely unknown. Methods: The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were used to analyze expression of glycolytic genes and clinical relevance in TNBC patients. Immunohistochemistry assay was performed to confirm the expression of glycolytic gene in breast cancer tissues. Gain-of-function and loss-of-function studies were conducted in TNBC cell lines. The effect of inflammatory microenvironment was carried out by coculture of macrophages and TNBC cells, and the expression of inflammatory cytokines were performed by real-time PCR. Results: We compared the differential genes expression in TNBC and non-TNBC by in silico analysis. Interestingly, the expression of glucose transporter 3 (Glut3) is upregulated in TNBC patients, compared to non-TNBC patients. Mechanistically, overexpression of Glut3 regulated the expression of epithelial-mesenchymal transition (EMT) genes and promoted invasiveness and metastasis of TNBC cells. Activation of IL6/ STAT3 signaling regulates the expression of Glut3 and glycolytic genes, and coexpression of IL6/Glut3 rendered poorer survival outcome, particularly in TNBC. Moreover, conditioned medium (CM) from Glut3-expressing tumor cells induced macrophage activation and production of pro-inflammatory cytokines, and patients with high Glut3 level associated with inflammaotry signautres. Conclusions: Upregulation of IL6/STAT3 signaling axis promotes expression of Glut3 and glycolytic genes. Elevation of Glut3 in TNBC induces macrophage activation and production of pro-inflammatory cytokines. Our data show the possible association between glucose metabolism and inflammatory microenvironment in TNBC. Legal entity responsible for the study: The author. Funding: Ministry of Science and Technology, Taiwan. Disclosure: The author has declared no conflicts of interest.
18P
Anti-cancer effects of differentiation-inducing factor-1 in triple negative breast cancer
F. Tetsuo1, M. Arioka2, F.Y. Takahashi3, F. Nishimura1, T. Sasaguri2 Dental, Kyushu University, Fukuoka, Japan, 2Clinical Pharmacology, Kyushu University, Fukuoka, Japan, 3Pharmacology, University of Occupational and Environmental Health, Kitakyushu, Japan
1
Background: Breast cancer is the most common cancer in women and its metastasis markedly exacerbates patients’ prognosis. In particular, triple negative breast cancer (TNBC), which lacks estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor 2 (HER2), is an aggressive subtype of breast cancer frequently forming metastatic lesions. Differentiation-inducing factor-1 (DIF-1) identified in Dictyostelium discoideum inhibits cell proliferation and migration of various mammalian cancer cells. However, the effect of DIF-1 on TNBC has not been examined. Here, we investigated whether DIF-1 shows anti-proliferative and anti-metastatic effects on TNBC by in-vivo and in-vitro experiments. Methods: In in-vivo experiments, we used cancer xenograft model mice in which murine TNBC 4T1/Luc cells (1.0 106 cells/mL) were injected into mammary fat pads to evaluate the effects of intragastric administration of DIF-1 (300 mg/kg/day) on the primary tumor growth and lung metastasis. In in-vitro experiments, we carried out assays for cell proliferation, migration and invasion to evaluate the anti-proliferative and antimetastatic effects of DIF-1. We also conducted Western blotting and real-time RT-PCR to identify the mechanisms for DIF-1’s anti-cancer effects. Results: In vivo, administration of DIF-1 significantly suppressed the primary tumor growth and lung metastasis without adverse effects such as weight loss and myelosuppression. In vitro, DIF-1 reduced cyclin D1 and c-Myc by inhibiting transcription and promoting degradation of the proteins, which resulted in the suppression of cell proliferation. DIF-1 also suppressed cell migration and invasion and reduced the protein
doi:10.1093/annonc/mdz238 | v5
Downloaded from https://academic.oup.com/annonc/article-abstract/30/Supplement_5/mdz238.016/5576344/ by guest on 07 October 2019
15P
it is presumed that they do not recognize the antigen, that is, they are in a state of immune tolerance and thus have a poor prognosis. In the group with granzyme B-high, the patients treated with anthracycline as adjuvant chemotherapy significantly improved their prognosis (p ¼ 0.043). The high expression of granzyme B may be a predictor of postoperative chemotherapy. Conclusions: Granzyme B is an important substance of cytotoxic pathway and has been possible to be an indicator of TIL activation. Legal entity responsible for the study: Masafumi Nakamura. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.