E0771 Cells Research Articles

Abstract 622: Progesterone promotes immunosuppression in the mammary gland through regulatory T cell activity

Abstract Estrogen and progesterone signaling together drive hormone-receptor positive breast cancer growth and progression. However, there have been limited studies on the independent role that progesterone and its receptor, the progesterone receptor, play in breast cancer development. Studies in our lab have shown that progesterone signaling promotes mammary tumor growth and changes the immune landscape of the mammary gland towards immunosuppression. Regulatory T-cells (Tregs) are immune cells known to promote immunosuppression in the tumor microenvironment, in part through decreasing the activity of anti-tumor CD8+ T cells. The data from our lab also show that progesterone increases the number of Tregs in the normal mammary gland. To determine if progesterone signaling increases the number of Tregs in the tumor-bearing mammary gland, we used a syngeneic orthotopic murine tumor model, E0771 cells, engineered to express mouse PR or control empty vector. Mice were treated with progesterone or placebo for 7 days before implantation of the modified E0771 cells. Results from this study show that progesterone promoted tumor growth, as well as changed the immune landscape of the tumor. Flow cytometry analysis at tumor endpoint showed that progesterone receptor-positive tumors treated with progesterone harbored more Tregs than placebo treated mice. Additionally, initial studies into activity markers harbored by Tregs in progesterone-treated mice indicate increased Treg activity following progesterone treatment. As progesterone treatment promoted tumor growth and Treg recruitment in progesterone receptor-positive tumors, these data imply that progesterone treatment may promote the release of secreted factors impacting the immune cells in the mammary microenvironment. To determine if progesterone treatment results in cytokine or chemokine changes that may affect Treg recruitment, cytokine arrays and enzyme-linked immunosorbent assays (ELISAs) were performed using cell culture supernatant from murine mammary tumor cells expressing progesterone receptor (E0771-PR) treated with progesterone (0-72 hours). E0771-PR cells treated with progesterone showed higher levels of cytokines and chemokines that have been linked to Treg recruitment and differentiation, such as CCL5. Our findings demonstrate that progesterone signaling promotes an immune suppressed microenvironment in the mammary gland through an increase in Tregs. This evidence provides rationale to investigate the use of anti-progestins as a mechanism to overcome the immune-cold tumor microenvironment seen in progesterone receptor positive breast tumors. Citation Format: Eilidh I. Chowanec, Lauryn R. Werner, Christy Hagan. Progesterone promotes immunosuppression in the mammary gland through regulatory T cell activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 622.

Abstract 2517: Optimizing breast cancer therapy by inhibiting the adenosine receptor and oxygen consumption

Abstract Immune checkpoint blockade has shown remarkable promise in melanoma and other tumor types. However, a large proportion of patients do not respond or develop acquired resistance. Tumors can activate multiple checkpoints and immunosuppressive pathways to evade immune surveillance, which are likely a cause of treatment failure. Therefore, inhibition of multiple checkpoints may be necessary for maximal efficacy of immunotherapies. The adenosine A2 receptor (A2aR), was shown to function as an immune checkpoint. Its blockade inhibited tumor growth and metastases and synergized with other checkpoint inhibitors. We propose that using AZD4635, a potent and selective A2aR antagonist, will enhance the therapeutic efficacy of anti-PD-1/PD-L1. In addition, we propose that blocking tumor oxygen consumption using deferiprone (DFP), phenformin (Phen) and metformin (Met) will further enhance A2aR and PD-1/PD-L1 blockade efficacy. We evaluated the effects of MET, Phen, DFP, and AZD4635 on tumor cells, T cell, and macrophage proliferation and effector function in vitro. IC50 measurements showed that T cells are the most sensitive to inhibition by these drugs, while 4T1 tumor cells appear to be the least sensitive. Naïve mouse T cells were activated with anti-CD3 and anti-CD28 coated dynabeads in the presence of titrating doses of all four drugs for 72 hours then analyzed by flow cytometry for proliferation and activation status. AZD4635 and DFP increased the expression of the activation marker CD25 (IL2Ra) on CD8 T cells. In addition, AZD4635 also increased expression of Granzyme B on both CD4 and CD8 T cells. Met appears to have little to no effect on T cell proliferation activation. We utilized the Seahorse XF Mito Stress Test assay to measure the mitochondrial respiratory activity of T cells in the presence these drugs. T cells were activated with dynabeads for 72 hours and then incubated with titrating doses of the drugs overnight before running on the Seahorse assay. DFP and Phen had the strongest effect on Maximal Respiration and Spare Respiratory Capacity at dilutions of 1uM followed by 16uM. A similar, albeit less profound effect was observed in groups treated with Met and AZD4635. Lastly, we obtained and generated two mouse breast cancer cell lines (4T1 and E0771) bearing HIF-1 reporter systems. We treated 4T1-HRE and E0771 cells with increasing doses of cobalt chloride (CoCl2) to chemically mimic hypoxia by inducing HIF1a expression. Both 4T1-HRE and E0771-HRE demonstrated increased luciferase activity that correlated with increasing doses of CoCl2 in vitro. Future experiments will focus on characterizing the adenosine pathway in vivo in 4T1-HRE and E0771-HRE tumors and examine how drugs that target the adenosine A2AaR receptor (AZD4635), and oxygen consumption (DFP, Phen and Met) influence tumor oxygen consumption in vivo as well as the activation states of immune cells in the tumor microenvironment. Citation Format: Sadna Budhu, Mayuresh Mane, Mamadou A. Bah, Juan Zurita, Inna Serganova, Soe Min, Anais Assouvie, Jedd D. Wolchok, Jason Koutcher, Vladimir Ponomarev, Taha Merghoub. Optimizing breast cancer therapy by inhibiting the adenosine receptor and oxygen consumption [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2517.

Abstract 6786: NT-I7 as an adjuvant to DNA neoantigen vaccination enhances and prolongs neoantigen-specific anti-tumor immunity

Abstract Background: IL-7 is an essential cytokine in the development and maintenance of antigen-specific T cells. NT-I7 (efineptakin alfa; NeoImmuneTech, Inc, Rockville, MD), is a long acting IL-7 composed of recombinant human IL-7 (rhIL-7) fused to a hybrid Fc antibody platform that has significantly improved in vivo stability and half-life compared to rhIL-7. DNA cancer neoantigen vaccines are a promising personalized cancer immunotherapy capable of generating strong anti-tumor immunity, but limited by a short-lived neoantigen-specific T cell response. We hypothesize NT-I7 as an adjuvant to a DNA neoantigen vaccine will enhance the magnitude and duration of neoantigen-specific anti-tumor immunity. Methods: C57BL/6 mice were vaccinated with DNA vaccine encoding neoantigens present in E0771 murine breast cancer model on days 0, 3, and 6. NT-I7 (5 mg/kg) was administered intravenously on day 4 or 13 during T cell expansion or contraction phase respectively. Primary T cell responses to neoantigens were assessed by IFN-γ ELISPOT. Tumor protection was evaluated by inoculating mice with 5×105 E0771 cells subcutaneously after receiving DNA vaccine with or without NT-I7. Duration of neoantigen-specific CD8 T cell immunity was determined by in vivo CFSE killing assay. Results: NT-I7 administered during the T cell contraction phase following the DNA vaccine increases the magnitude of neoantigen-specific T cell responses on day 20 compared to DNA vaccine alone. DNA vaccination and NT-I7 alone prior to tumor inoculation confers partial protection against tumor growth compared to control while DNA + NT-I7 during contraction phase rejects 100% of tumors. Compared to DNA vaccine alone, DNA vaccine + NT-I7 during contraction phase induces greater neoantigen-specific killing on day 41 (34.6% vs. 11.97%, p=0.03). Conclusion: NT-I7 as an adjuvant to a DNA neoantigen vaccine increases the magnitude and duration of neoantigen-specific anti-tumor immunity in a murine model. Clinical application of NT-I7 may help overcome immunologic shortcomings of current neoantigen vaccines. Citation Format: Michael Y. Chen, Ina Chen, Suangson Supabphol, Alexandra A. Wolfarth, Sara F. Martinez, Byung Ha Lee, Lijin Li, Xiuli Zhang, Peter S. Goedgebuure, William E. Gillanders. NT-I7 as an adjuvant to DNA neoantigen vaccination enhances and prolongs neoantigen-specific anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6786.

Abstract P3-06-05: Effects of isoflavone genistein, combined with anti-estrogen fulvestrant, on anti-PD1 therapy response against E0771 mammary tumors in mice

Abstract Although immune checkpoint blockers (ICBs) have revolutionized the treatment of many cancers, only less than 30% of patients respond to any ICB as a monotherapy. Estrogens, produced by the adipose tissue in the mammary glands, are immunomodulators in the tumor microenvironment (TME). Estrogens suppress anti-tumor immunity and activate immunosuppression through the estrogen receptor α (ERα) in the immune cells but activate anti-tumor immunity through ERβ. Genistein (GEN) is a biologically active isoflavone and is structurally similar to estradiol (E2). However, in contrast to E2, GEN binds preferentially to ERβ. Here we studied if GEN combined with ERα inhibitor fulvestrant (Fulv) alters response to anti-PD1 therapy in ERα negative, ERβ positive E0771 mouse mammary tumors. Syngeneic C57BL/6 mice were fed either a control (CON) diet or GEN supplemented diet for 5 weeks and then allografted E0771 mammary tumor cell into the 4th mammary fat pad. When tumors reached an average size of 16mm2, mice in both diet groups were further divided into 4 sub-groups treated with: 1) anti-PD1 (150µg or 100µg every 3 days for a total of 3 doses), 2) Fulv (1mg) once weekly, 3) anti-PD1+Fulv or 4) IgG control group. We found that three doses of 150µg of anti-PD1 alone effectively reduced tumor growth in the CON (p=0.0005) group and its efficacy was notably weakened in the GEN group (p=0.04).. Combination with Fulv did not improve PD1 response. These findings suggest that when using a highly effective anti-PD1 dose, GEN may reduce rather than improve the response, and anti-ERα does not add to anti-PD1’s effect in control of GEN group. A suboptimal dose (100 µg) of anti-PD1 alone did not inhibit E0771 tumor growth in mice fed CON diet, but in the GEN group the tumor inhibition by anti-PD1 alone almost reached statistical significance (p=0.057). The combination of suboptimal PD1+Fulv significantly reduced tumor growth compared with IgG (p=0.04) or Fulv (p=0.02). During GEN feeding, anti-PD1+Fulv was even more potent in inhibiting tumor growth when compared with IgG group (p=0.02) and Fulv group (p=0.007). Thus, when anti-PD1 alone is not effective, either Fulv or GEN will improve responsiveness to anti-PD1. In the suboptimal anti-PD1 dose experiment, GEN feeding increased CD8+ T cells (P=0.04) and decreased CD4+Foxp3+ cells (p=0.01) in the TME. Further, GEN reduced the proportion of immunosuppressive MDSC cells (CD3-CD11B+Ly6Ghigh6Clow) in the PD1+ Fulv group (p=0.01) in the Peyer’s patches. In vitro experiments showed that both GEN and Fulv, and especially their combination, inhibited the E0771 cells proliferation, indicating that these two treatments can also directly suppress the growth of ERα negative but ERβ positive breast cancer cells. Taken together, the data show that anti-estrogens and GEN in the diet may improve response to anti-PD1 therapy, possibly both through activation of tumor immune response and inhibition of tumor cell proliferation and induction of tumor cell death. Citation Format: Fabia de Oliveira Andrade, Vivek Verma, Ester Molina Hoyo, Karla Andrade de Oliveira, Pal Koak, Lu Jin, Leena Hilakivi-Clarke. Effects of isoflavone genistein, combined with anti-estrogen fulvestrant, on anti-PD1 therapy response against E0771 mammary tumors in mice [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-06-05.

NSC243928 Treatment Induces Anti-Tumor Immune Response in Mouse Mammary Tumor Models.

NSC243928 induces cell death in triple-negative breast cancer cells in a LY6K-dependent manner. NSC243928 has been reported as an anti-cancer agent in the NCI small molecule library. The molecular mechanism of NSC243928 as an anti-cancer agent in the treatment of tumor growth in the syngeneic mouse model has not been established. With the success of immunotherapies, novel anti-cancer drugs that may elicit an anti-tumor immune response are of high interest in the development of novel drugs to treat solid cancer. Thus, we focused on studying whether NSC243928 may elicit an anti-tumor immune response in the in vivo mammary tumor models of 4T1 and E0771. We observed that NSC243928 induced immunogenic cell death in 4T1 and E0771 cells. Furthermore, NSC243928 mounted an anti-tumor immune response by increasing immune cells such as patrolling monocytes, NKT cells, B1 cells, and decreasing PMN MDSCs in vivo. Further studies are required to understand the exact mechanism of NSC243928 action in inducing an anti-tumor immune response in vivo, which can be used to determine a molecular signature associated with NSC243928 efficacy. NSC243928 may be a good target for future immuno-oncology drug development for breast cancer.

Abstract IA046: Mechanistic targets and strategies to break the obesity-cancer link

Abstract Obesity promotes the incidence and progression of many cancers including breast cancer. Mechanisms underpinning obesity-driven breast cancer that could be harnessed to intercept this deleterious relationship are not completely understood. Here we apply multi-omics analysis of tumor and adipose to delineate candidate mediators underlying the procancer effects of obesity, and anticancer effects of weight loss, in a mouse model of obesity-driven breast cancer. We modeled bariatric surgery and nonsurgical weight loss in obese C57BL/6NCrl mice fed a high-fat diet (60 kcal% fat) using vertical sleeve gastrectomy or a sham surgery paired with (i) dietary weight loss via ad libitum low-fat diet (10 kcal% fat), (ii) 30% chronic calorie restriction, or (iii) intermittent calorie restriction. Once weight loss stabilized, mammary tumors were induced by orthotopic transplantation of E0771 cells, a model of triple negative breast cancer (TNBC). While bariatric surgery and a switch to low-fat diet were each moderately effective at reducing tumor mass, chronic and intermittent calorie restriction regimens were each highly effective in reducing tumor burden. Tumor transcriptomic analysis using gene set enrichment analysis revealed that weight loss interventions reversed obesity-associated immunosuppression. Mediation analysis identified weight loss and fat mass loss as significant mediators of the reduced tumor mass seen in all interventions. Integration of adipose transcriptomics and reduced-representation bisulfite sequencing identified NFKB1, FXR1, and BACH1 as regulators overrepresented in differentially methylated genes, and conserved with predicted regulators of transcriptional remodeling in human adipose tissue following bariatric surgery. These data indicate that weight loss through dietary and surgical means promotes antitumor immunity, in part via remodeling of tumor adjacent adipose tissue. We have identified several candidate mediators of adipose remodeling that may underpin the antitumor effects of bariatric surgery, and these mediators represent potential mechanism-based targets for future interventions. Citation Format: Michael F. Coleman. Mechanistic targets and strategies to break the obesity-cancer link [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr IA046.

Preclinical Platform Using a Triple-negative Breast Cancer Syngeneic Murine Model to Evaluate Immune Checkpoint Inhibitors.

To evaluate the feasibility of syngeneic mouse models of breast cancer by analyzing the efficacy of immune checkpoint inhibitors (ICIs) and potential predictive biomarkers. To establish the murine triple-negative breast cancer (TNBC) models, JC, 4T1, EMT6, and E0771 cells were subcutaneously implanted into female syngeneic mice. When the tumor reached 50-100 mm3, each mouse model was divided into a treatment (using a murine PD-1 antibody) and a no-treatment control group. The treatment group was further divided into the responder and non-responder groups. Potential predictive biomarkers were evaluated by analyzing serum cytokines, peripheral blood T cells and tumor infiltrating immune cells. The EMT6 model showed the highest tumor response rate (54%, 6/11) of the syngeneic models: 4T1 (45%, 5/11), JC (40%, 4/10), or E0771 (23%, 3/13). Early changes in tumor size at 7 days post-PD-1 inhibitor treatment predicted the final efficacy of the PD-1 inhibitor. Peripheral blood CD8+ and CD4+ T cells with or without Ki67 expression at 7 days post-PD-1 inhibitor treatment were higher in the finally designated responder group than in the non-responder group. At the time of sacrifice, analyses of tumor infiltrating lymphocytes consistently supported these results. We also demonstrated that retro-orbital blood sampling procedures (baseline, 7 days post-treatment, time of sacrifice) were safe for serum cytokine analyses, suggesting that our preclinical platform may be used for biomarker research using serum cytokines. Our syngeneic mouse model of TNBC is a feasible preclinical platform to evaluate ICI efficacy combined with other drugs and predictive biomarkers in the screening process of immune-oncology drug development.

Steroid receptor coactivator-3 inhibition generates breast cancer antitumor immune microenvironment

BackgroundThe tumor immune microenvironment (TIME) generated by cancer-infiltrating immune cells has a crucial role in promoting or suppressing breast cancer progression. However, whether the steroid receptor coactivator-3 (SRC-3) modulates TIME to progress breast cancer is unclear. Therefore, the present study evaluates whether SRC-3 generates a tumor-promoting TIME in breast tumors using a syngeneic immune-intact mouse model of breast cancer.MethodsWe employed E0771 and 4T1 breast cancer in immune-intact syngeneic female C57BL/6 and BALB/c mice, respectively. SI-2, a specific small-molecule inhibitor of SRC-3, was administered daily (2.5 mg/kg) to E0771 and 4T1 breast tumor-bearing immune-intact mice. In addition, SRC-3 knockdown (KD)-E0771 and SRC-3 KD-4T1 cells and their parental breast cancer cells were injected into their syngeneic immune-intact female mice versus immune-deficiency mice to validate that the host immune system is required for breast tumor suppression by SRC-3 KD in immune-intact mice. Furthermore, tumor-infiltrating immune cells (such as CD4+, CD8+, CD56+, and Foxp3+ cells) in E0771 and 4T1 breast cancers treated with SI-2 and in SRC-3 KD E0771 and 4T1 breast cancers were determined by immunohistochemistry. Additionally, cytokine levels in SI-2-treated and SRC-3 KD E0771 breast tumors and their control cancers were defined with a Mouse Cytokine Array.ResultsSRC-3 inhibition by SI-2 significantly suppressed the progression of breast cancer cells (E0771 and 4T1) into breast cancers in immune-intact syngeneic female mice. SRC-3 KD-E0771 and -4T1 breast cancer cells did not produce well-developed tumors in immune-intact syngeneic female mice compared to their parental cells, but SRC-3 KD breast cancers were well developed in immune-defective host mice. SRC-3 inhibition by SI-2 and SRC-3 KD effectively increased the numbers of cytotoxic immune cells, such as CD4+ and CD8+ T cells and CD56+ NK cells, and Interferon γ (Ifng) in breast cancers compared to vehicle. However, SI-2 treatment reduced the number of tumor-infiltrating CD4+/Foxp3+ regulatory T (Treg) cells compared to vehicle treatment. In addition, SRC-3 inhibition by SI-2 and SRC-3 KD increased C-X-C motif chemokine ligand 9 (Cxcl9) expression in breast cancer to recruit C-X-C motif chemokine receptor 3 (Cxcr3)-expressing cytotoxic immune cells into breast tumors.ConclusionsSRC-3 is a critical immunomodulator in breast cancer, generating a protumor immune microenvironment. SRC-3 inhibition by SI-2 or SRC-3 KD activates the Cxcl9/Cxcr3 axis in breast tumors and enhances the antitumor immune microenvironment to suppress breast cancer progression.

Intravascular Administration of Acridine Orange and Zoledronate in a Bone Metastasis Model of Breast Cancer.

This study evaluated the effect of haematogenous administration of acridine orange (AO) alone and in combination with zoledronate (ZOL) on bone metastases. E0771 cells (1.0×105 cells/10 μl) were injected directly into the right femur of female mice. The mice were divided into five groups according to treatment (drugs and irradiation) and were reared and sacrificed after 6 weeks. Micro-computed tomography (μCT) was performed to calculate the destruction rate of the femur bone. We measured tumour weight and volume at sacrifice and performed terminal deoxynucleotidyl transferase dUTP Nick-End Labelling staining of tumours. At 4 weeks, the bone destruction rate was lower in the AO+ZOL group than in the radiation group. At 6 weeks, the AO+ZOL group had a lower bone destruction rate than the control and radiation groups; the ZOL group had a lower rate than the radiation group. The AO and AO+ZOL groups had suppressed tumour weight and volume compared to the control and radiation groups. The number of extraosseous apoptotic cells was higher in the AO+ZOL group than in all other groups except the AO group. In a model of local bone metastasis of breast cancer, haematogenous administration of AO reduced tumour size and more so when combined with ZOL.

Novel Methods of Targeting IL-1 Signalling for the Treatment of Breast Cancer Bone Metastasis.

Simple SummaryThe pro-inflammatory cytokine, IL1β, plays a pivotal role in breast cancer bone metastasis. Inhibiting IL-1 signalling with the IL1β specific antibody, Canakinumab, or the IL1R1 antagonist Anakinra almost eliminates bone metastases but has adverse effects on tumours growing outside of the bone and immune regulation. This current study demonstrated that pharmacological inhibition of other members of the IL-1 signalling pathway Caspase-1, IL1β and IL1R reduced migration and invasion of E0771 and Py8119 cells in vitro and also reduced spontaneous metastasis and metastatic outgrowth of breast cancer in the bone, in vivo. Interestingly, targeting IRAK1 had no anti-tumour effects. Importantly, inhibiting Caspase-1 reduces bone metastasis without adversely affecting tumours outside of bone or immune cell regulation, suggesting that targeting immediately upstream of IL1β may be a good therapeutic strategy for treating patients with breast-cancer-induced bone disease.Breast cancer bone metastasis is currently incurable. Evidence suggests that inhibiting IL-1 signalling with the IL1R antagonist, Anakinra, or the IL1β antibody, Canakinumab, prevents metastasis and almost eliminates breast cancer growth in the bone. However, these drugs increase primary tumour growth. We, therefore, investigated whether targeting other members of the IL-1 pathway (Caspase-1, IL1β or IRAK1) could reduce bone metastases without increasing tumour growth outside of the bone. Inhibition of IL-1 via MLX01 (IL1β secretion inhibitor), VRT043198/VX765 (Caspase-1 inhibitor), Pacritinib (IRAK1 inhibitor) or Anakinra (IL1R antagonist) on tumour cell viability, migration and invasion were assessed in mouse mammary E0771 and Py8119 cells in vitro and on primary tumour growth, spontaneous metastasis and metastatic outgrowth in vivo. In vitro, Inhibition of IL-1 signalling by MLX01, VRT043198 and Anakinra reduced migration of E0771 and Py8119 cells and reversed tumour-derived IL1β induced-increased invasion and migration towards bone cells. In vivo, VX765 and Anakinra significantly reduced spontaneous metastasis and metastatic outgrowth in the bone, whereas MLX01 reduced primary tumour growth and bone metastasis. Pacritinib had no effect on metastasis in vitro or in vivo. Targeting IL-1 signalling with small molecule inhibitors may provide a new therapeutic strategy for breast cancer bone metastasis.

ICAM-1 on Breast Cancer Cells Suppresses Lung Metastasis but Is Dispensable for Tumor Growth and Killing by Cytotoxic T Cells.

Breast tumors and their derived circulating cancer cells express the leukocyte β2 integrin ligand Intercellular adhesion molecule 1 (ICAM-1). We found that elevated ICAM-1 expression in breast cancer cells results in a favorable outcome and prolonged survival of breast cancer patients. We therefore assessed the direct in vivo contribution of ICAM-1 expressed by breast cancer cells to breast tumorigenesis and lung metastasis in syngeneic immunocompetent mice hosts using spontaneous and experimental models of the lung metastasis of the C57BL/6-derived E0771 cell line, a luminal B breast cancer subtype. Notably, the presence of ICAM-1 on E0771 did not alter tumor growth or the leukocyte composition in the tumor microenvironment. Interestingly, the elimination of Tregs led to the rapid killing of primary tumor cells independently of tumor ICAM-1 expression. The in vivo elimination of a primary E0771 tumor expressing the ovalbumin (OVA) model neoantigen by the OVA-specific OVA-tcr-I mice (OT-I) transgenic cytotoxic T lymphocytes (CTLs) also took place normally in the absence of ICAM-1 expression by E0771 breast cancer target cells. The whole lung imaging of these cells by light sheet microscopy (LSM) revealed that both Wild type (WT)- and ICAM-1-deficient E0771 cells were equally disseminated from resected tumors and accumulated inside the lung vasculature at similar magnitudes. ICAM-1-deficient breast cancer cells developed, however, much larger metastatic lesions than their control counterparts. Strikingly, the vast majority of these cells gave rise to intravascular tumor colonies both in spontaneous and experimental metastasis models. In the latter model, ICAM-1 expressing E0771- but not their ICAM-1-deficient counterparts were highly susceptible to elimination by neutrophils adoptively transferred from E0771 tumor-bearing donor mice. Ex vivo, neutrophils derived from tumor-bearing mice also killed cultured E0771 cells via ICAM-1-dependent interactions. Collectively, our results are a first indication that ICAM-1 expressed by metastatic breast cancer cells that expand inside the lung vasculature is involved in innate rather than in adaptive cancer cell killing. This is also a first indication that the breast tumor expression of ICAM-1 is not required for CTL-mediated killing but can function as a suppressor of intravascular breast cancer metastasis to lungs.

Adropin Improves Radiation-Induced Myocardial Injury via VEGFR2/PI3K/Akt Pathway.

Mediastinal cancer radiotherapy exposes the heart and causes myocardial injury. It is of utmost importance to identify effective prevention and treatment targets. In this study, the regulatory role of adropin (Ad) in radiation-induced myocardial injury (RIMI) was explored in mice. After C57BL/6 mice were administered E0771 cells and received radiotherapy, the effects of exogenous Ad intervention on myocardial fibrosis, apoptosis, microvessel density, oxidative stress, and protein expression levels were observed. The results showed that exogenous Ad effectively improved cardiac function, suppressed oxidative stress, inhibited myocardial fibrosis, reduced myocardial apoptosis, and promoted microangiogenesis in RIMI mice. Ad also downregulated the expression levels of transforming growth factor β1 (TGF-β1), NADPH oxidase 4 (NOX4), and cleaved caspase 3 and upregulated the expression of phosphor-endothelial nitric oxide synthase (p-eNOS). However, the above-mentioned effects of Ad were significantly reversed in Ad−/− mice. Radiotherapy resulted in the downregulation of phosphor-vascular endothelial growth factor receptor (p-VEGFR2) and p-Akt in myocardial tissue, which were upregulated by Ad. However, after targeted inhibition of VEGFR2 with apatinib, the effect of Ad on improving RIMI was significantly reversed. Taken together, exogenous Ad significantly ameliorated RIMI by reducing oxidative stress, promoting microangiogenesis, and inhibiting myocardial fibrosis and apoptosis. The underlying molecular mechanism involved may be elucidated by activation of the VEGFR2/PI3K/Akt pathway.

Abstract 1350: The role of phosphatidylserine scramblases in tumor progression

Abstract Introduction: Phosphatidylserine (PS), an anionic phospholipid, is asymmetrically distributed in the inner leaflet of the phospholipid bilayer in normal cells under homeostasis. However, PS is constitutively externalized in the tumor microenvironment (TME); on viable cells that undergo stress, such as hypoxic tumor cells, stressed tumor vascular endothelial cells, as well as on cells undergoing apoptosis. This high PS harbors a pro-tumorigenic micro-environment, as myeloid cells expressing PS receptors (Mertk, Tyro3 and Axl) engage with externalized PS and secrete immunosuppressive cytokines. PS is externalized on viable cells by a calcium activated scramblase TMEM16F and on apoptotic cells by a caspase activated scramblase called Xkr8. Here, to study the mechanisms by which PS is externalized in the TME, we hypothesize that TMEM16F and Xkr8 are the major scramblases responsible for creating an immune suppressive milieu in the tumor microenvironment. Methods: To study the mechanisms of PS dysregulation in the tumor microenvironment, TMEM16F and Xkr8 were knocked out in E0771 murine breast cancer cell line using CRISPR/Cas9. The knockouts were validated by western blotting, q-RT-PCR and surveyor assays. Results: TMEM16F KO E0771 cells express lower basal levels of PS compared to WT E0771 cells when detected by Annexin V staining by flow cytometry. TMEM16F KO cells are also less responsive to activation by calcium ionophore. TMEM16F KO, Xkr8 KO and WT E0771 cells have similar viability and migration patterns in response to Gas6, indicating that the cells themselves do not have altered oncogenic potential. TMEM16F KO E0771 cells injected into the inguinal mammary gland fat pad of C57BL6 syngeneic mice showed modest but significantly reduced tumor growth compared to the E0771 WT tumors, suggesting that TMEM16F mediated PS externalization could play a role in exacerbating tumor progression. Conclusion: TMEM16F driven PS externalization contributes to tumor growth. The effect of TMEM16F KO on immune regulation of the tumor milieu will be discussed. These data corroborate previous studies targeting PS in tumors therapeutically using anti-PS antibodies and suggests a role for scramblases in pleiotropic immune-suppression in the tumor. Citation Format: Varsha Gadiyar, Viralkumar Davra, David Calianese, Kevin Lahey, Raymond B. Birge. The role of phosphatidylserine scramblases in tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1350.

Abstract 1321: Effects of sulindac on inflammatory and cellular responses in mammary tumors from obese mice

Abstract Background: Breast cancer is the second leading cause of cancer-related death in US women. Obesity (BMI > 30 kg/m2) promotes breast cancer incidence, progression, and mortality in women of all ages. The prevalence of obesity in the US has increased dramatically over recent decades and continues to rise. Although obesity is associated with increased adipose tissue inflammation and metabolic dysfunction the exact mechanisms regulating obesity-exacerbated tumor growth are relatively unknown. Nonsteroidal anti-inflammatory drugs (NSAIDs), like sulindac, inhibit cyclooxygenase-1 and cyclooxygenase-2 and modulate the secretion of pro-inflammatory cytokines. The purpose of this study was to investigate the mechanisms underlying the anticancer effects of sulindac in our E0771 mouse model of obesity and mammary tumor growth. Methods: C57BL/6J mice were fed either a high-fat diet (n=16) or a low-fat control diet (n=12) for 15 weeks. Half of the control (n=6) and DIO (n=8) mice were then treated with 140 ppm sulindac in their respective diets for 4 weeks prior to orthotopic injection of 20,000 E0771 cells. Tumors were harvested and weighed, and serum was collected 4 weeks following injection. Bulk transcriptional profile analysis was performed on E0771 tumors from untreated control and DIO mice relative to sulindac-treated control and DIO mice using Affymetrix microarrays followed by gene set enrichment analysis (GSEA) to identify significantly enriched gene sets. Serum cytokine levels were measured in all mice using the Bio-Plex Pro mouse chemokine panel 33-plex; multiple linear regression was conducted to determine cytokines associated with mammary tumor size, independent of diet or treatment group assignment. Results: DIO mice had increased tumor weight relative to control mice, while sulindac decreased tumor weight in DIO but not control mice. GSEA analysis revealed only one gene set upregulated in the DIO tumors relative to control or DIO sulindac tumors. Immune-related gene sets were commonly enriched in tumors from control and DIO sulindac relative to DIO mice, suggesting that sulindac restored immune signaling in tumors from DIO mice. Leading edge analysis of the significantly enriched gene sets showed that: 1) the reversal of DIO-mediated immunosuppression by sulindac occurred via principally overlapping genes; and 2) cytokine and immune genes predominate the transcriptional effects of sulindac treatment in DIO mice. Sulindac also modified circulating serum cytokine levels, but multiple linear regression showed that cytokine levels were not independently predictive of tumor weight in our study. Conclusions: The study suggests that sulindac can reverse transcriptional changes in mammary tumors from obese mice and restore immunosurveillance. Sulindac, or potentially other NSAIDs, may protect against obesity–driven mammary tumor growth via remodeling of antitumor immune function. Citation Format: Morgan E. Cody, Shannon B. McDonell, Qing Shi, Elaine M. Glenny, Michael F. Coleman, Stephen D. Hursting. Effects of sulindac on inflammatory and cellular responses in mammary tumors from obese mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1321.

Abstract 2050: Treatment with anti-PD-1 monoclonal antibody increases levels of MIP-1α and other cytokines, and suppresses tumor growth in a mouse model of triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC), characterized by the lack of estrogen and progesterone receptors and HER2, is an aggressive form of breast cancer that accounts for 15-20% of all diagnosed cases. There are few targeted therapies available for TNBC, but immune checkpoint inhibitors (ICIs) such as Pembrolizumab have been shown to be effective in a subset of TNBC patients. However, because ICIs are rarely administered as a monotherapy, there remains little foundational understanding of how single-agent immunotherapies alter the immune landscape and which circulating immune profiles predict therapeutic response in TNBC. Therefore, further investigation into the distinct immune signatures of ICIs are necessary for developing a rational basis for selecting optimal treatment regimens for TNBC. Methods: E0771 cells, a C57Bl/6 mouse-derived TNBC cell line, were injected into the bilateral mammary pads of 8 week old female C57Bl/6J mice. Control mice were injected with saline into mammary pads. After tumors developed (14 days post-implantation), E0771 mice received 200 ug doses of immunotherapy (anti-PD-1, anti-LAG-3, or anti-TIM-3 antibodies) or IgG isotype control every four days. After 3 doses, treatments were stopped and serum was collected from “on-treatment” mice. Fourteen days after the third treatment, serum was collected from “post-treatment” mice. A panel of 31 cytokines, chemokines, and growth factors were analyzed in “pre-treatment”, “on-treatment”, “post-treatment”, and saline control mouse serum using a BioPlex 200 multiplex system (Eve Technologies). Results: Mice treated with anti-PD-1 antibody showed significantly higher levels of several cytokines and chemokines compared to isotype control mice. Among the most significantly increased cytokines in the post-treatment anti-PD-1 group was macrophage inflammatory protein-1α (MIP-1α), or CCL3. MIP-1α is a chemotactic cytokine secreted by several types of immune cells that is known to attract macrophages and CD8+ T cells. More subtle changes in levels of several cytokines, chemokines, and growth factors were observed in anti-LAG-3 and anti-TIM-3 treatment groups. At the post-treatment timepoint, mice treated with anti-PD-1 antibody had smaller tumor sizes than mice treated with anti-LAG-3 and anti-TIM-3 antibodies, indicating that PD-1 blockade slows tumor growth and disease progression. Conclusion: Anti-PD-1 treatment induced more distinct circulating immune profiles and was more effective at controlling tumor growth than anti-LAG-3 and anti-TIM-3 treatments. These results may also suggest MIP-1α-mediated tumor regression in response to anti-PD-1 treatment in TNBC. Citation Format: Payton De La Cruz, Kathryn Grive, Jennifer Ribeiro. Treatment with anti-PD-1 monoclonal antibody increases levels of MIP-1α and other cytokines, and suppresses tumor growth in a mouse model of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2050.

Abstract 3010: Targeting metabolic pathways through pharmacological and chemotherapeutic interventions to improve triple-negative breast cancer therapy

Abstract Triple-negative breast cancers (TNBCs) lack targeted therapies, leaving surgery and systemic chemotherapy as current standard approaches for treatment. However, chemotherapy resistance is a major clinical challenge. Stimulation of receptor tyrosine kinases such as insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF-1R) activates the protein kinase A (PKA), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling cascades. Activation of these pathways is observed in the majority of TNBC tumors and promotes pro-proliferative signaling. Thus IGF-1R/IR are potential therapeutic targets in TNBC, but clinical trials of anti-IGF-1R/IR therapies have consistently shown minimal and often heterogeneous therapeutic responses. Our objective was to determine whether metabolic reprogramming achieved by combination regimens, specifically the IGF-1R/IR inhibitor BMS-754807 and/or treatment with the platinum-based chemotherapeutic agent, carboplatin, promotes autophagy induction and sensitizes cancer cells to autophagy inhibition. Cytotoxicity of BMS-754807, alone or in combination with carboplatin and/or the autophagy inhibitor, hydroxychloroquine, was assessed in murine (E0771, metM-Wntlung, B6C3TAg 2.51) and human (MDA-MB-231, MDA-MB-468) models of TNBC using MTT assays. Analysis of mitochondrial mass was measured in cells stained with MitoTracker Green FM using flow cytometry. Autophagic flux was measured with an mCherry-EGFP-LC3B tandem fluorescent protein. BMS-754807 (10μM) significantly induced 22-51% cytotoxicity in all cell lines tested, with E0771 cells showing the strongest response. Treatment with BMS-754807 (2.5µM) significantly increased mitochondrial mass (31%) compared to untreated cells. In addition, co-treatment with BMS-754807 and carboplatin further suppressed cell viability and regulated phosphorylation of the double-stranded DNA damage response proteins γ;-H2AX and Chk2. Co-treatment with BMS-754807 and carboplatin also altered the GFP/RFP ratio in LC3-expressing cells, suggesting modulation of autophagic flux. Finally, the addition of the autophagy inhibitor hydroxychloroquine further induced cytotoxicity when coupled with BMS-754807 and carboplatin. This work indicates that IGF-1R/IR inhibition remodels metabolism in TNBC cells, potentially synergizing with carboplatin to induce double stranded DNA damage. Moreover, the combination of IGF-1R/IR and carboplatin can further collaborate with autophagy inhibition to strongly suppress TNBC cell growth. We conclude that inhibiting nutrient-sensing metabolic pathways such as IGF-1R/IR in combination with chemotherapy and/or autophagy inhibition warrants additional study as a strategy to improve therapeutic responses in women with TNBC. Citation Format: Alyssa N. Ho, Violet A. Kiesel, Scott P. Connelly, Michael F. Coleman, Stephen D. Hursting. Targeting metabolic pathways through pharmacological and chemotherapeutic interventions to improve triple-negative breast cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3010.

Reversing the Genomic, Epigenetic, and Triple-Negative Breast Cancer-Enhancing Effects of Obesity.

Obesity is an established risk and progression factor for triple-negative breast cancer (TNBC). Given rising global rates of obesity and TNBC, strategies to reduce the burden of obesity-driven TNBC are urgently needed. We report the genomic, epigenetic, and procancer effects of obesity are reversible by various calorie restriction regimens.

457 Semaphorin7a expression in breast cancer promotes susceptibility to immune checkpoint blockade

OBJECTIVES/GOALS: The goal of this study is to decipher the mechanisms by which breast cancers evade the immune system to facilitate metastasis through the lymphatic vasculature. We believe this is caused, in part, by semaphorin 7A (SEMA7A) as its expression is associated with increased lymphatic vessels, tumor-associated macrophages, and metastasis in breast cancer. METHODS/STUDY POPULATION: We have observed that breast cancer cells, lymphatics, and macrophages exhibit SEMA7A-dependent expression of PD-L1 in vitro, which may make SEMA7A+ tumors more sensitive to anti-PD-1/PD-L1 immunotherapies. Therefore, we utilized multiple orthotopic, immunocompetent mouse models to determine if SEMA7A expression makes tumors more susceptible to immune checkpoint blockade. E0771 and 66cl4 mouse mammary carcinoma cells were injected orthotopically into the #4 mammary fat pads. Once tumors reached 50-100 mm^3 mice were injected intraperitoneally with 250ug of anti-PD-1, anti-PD-L1, or IgG control every third day. Tumors were measured with calipers every other day and harvested for flow cytometry to determine the effect of immune checkpoint blockade on the TME in SEMA7A+ tumors. RESULTS/ANTICIPATED RESULTS: We reveal that growth of SEMA7A overexpressing (OE) tumors, but not controls, was significantly slowed with both anti-PD-1 and anti-PD-L1 treatments. Flow cytometric analysis of cells from the TME revealed increased LECs, TAMs, and PoEMs in SEMA7A+ tumors, compared to controls – all populations had higher PD-L1 expression, which was decreased with both anti-PD-1 or anti-PD-L1. We also observed a decrease in PD-L1 expression on the tumor cells with treatment. Furthermore, LEC, TAM, and PoEM presence was decreased within the TME alongside increased presence of activated CD4 and CD8 T cells. Collectively, our results suggest that SEMA7A expression in breast cancers activates a major pathway associated with immune evasion and helps to establish a pro-tumor microenvironment. DISCUSSION/SIGNIFICANCE: SEMA7A expression establishes a pro-tumor microenvironment, which can be targeted with readily available FDA approved drugs such as immune checkpoint-based therapies. Since SEMA7A+ breast cancers have high rates of metastasis, more specific treatments for these patient populations should be explored.

Abstract PD3-08: Novel cancer stem cell inhibitor 108600 modulates tumor immunomicroenvironment of triple negative breast cancer (TNBC)

Abstract 108600, a novel CK2/DYRK1/TNIK inhibitor, targets and inhibits cancer stem cells (CSC) in triple negative breast cancer (TNBC), inhibiting tumor growth and metastases in patient-derived xenograft models. CSC’s have been shown to promote immune evasion of several types of cancer. Specifically, over-expression of CK2 has been shown to promote intratumoral recruitment of myeloid derived suppressor cells. We investigated the effects of 108600 treatment on the immune microenvironment of triple negative breast cancer since targeting CSC’s could promote favorable anti-tumor immune responses and mechanistically contribute to tumor growth inhibition. Methods:C57BL/6 mice bearing murine triple negative E0771 tumors were generated by orthotopic injection and treated either with vehicle control or 108600 for 5 days. Tumor growth was assessed by daily caliper measurement All tumors were recovered at endpoint, and processed for RNA sequencing, flow cytometry or Western blot analysis. Results:108600 treatment significantly inhibited growth of E0771 tumors in vivo, demonstrating for the first time efficacy of 108600 against TNBC in immunocompetent models. 108600 treatment decreased intratumoral phosphorylation and expression of 108600 targets AKT1 (Ser 129) and Cyclin D1, which are substrates of CK2α and Dyrk1, respectively. To further explore nature of transcriptional and signaling pathways affected by 108600 treatment in vivo, RNA sequencing was performed. DGE and subsequent annotation analysis showed that various immune (GO:0006955, GO) and inflammatory (GO:0006954, GO) signaling pathways associated with Stat1 (mmu04062, KEGG) and IFNγ (mmu04060, KEGG) were down-regulated in 108600-treated E0771 tumors. GSEA (Gene set enrichment analysis) indicated that the regulatory T cell (Treg) population (GSE42021, GSE40685) was suppressed by 108600 treatment. To validate these findings, tumor infiltrating leukocytes (TIL) were isolated from vehicle or 108600-treated tumors and analyzed by flow cytometry. CD4, CD25, and FOXP3 expression was used to gate the Treg population. The Treg population was significantly suppressed by 108600 treatment, confirming the RNA sequencing results. 108600 also likely regulates expression of immunomodulatory molecules in TNBC tumor cells since 108600 treatment increased PD-L1 surface expression on E0771 cells in vitro. Conclusions:Our study supports 108600, an inhibitor that targets breast cancer stem cells, as a modulator of the immune microenvironment of TNBC. 108600 suppresses the Treg population among the TIL population and increases tumoral expression of PD-L1. Tregs have been associated with disease progression and metastases of TNBC. Further studies are ongoing to clarify the functional consequences of these changes in the setting of tumor growth inhibition. Synergies between 108600 and checkpoint inhibition are also under investigation and could lead to novel combination therapies for TNBC. Citation Format: Katsutoshi Sato, Stacey J. Baker, E. P. Reddy, Hanna Y. Irie. Novel cancer stem cell inhibitor 108600 modulates tumor immunomicroenvironment of triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-08.

Abstract P1-06-02: Targeted deletion of Kindlin-2 in mouse mammary glands inhibits tumor growth, invasion and metastasis downstream of TGF-β/EGF oncogenic signaling pathway

Abstract Cancer metastasis is a complex process by which cancer cells migrate through the blood and the lymphatic systems to lodge and proliferate in distant sites and organs in the body. Metastasis is the main cause of death in patients suffering from cancer, including those patients with breast cancer. Breast cancer (BC) is the most frequently diagnosed malignancy in women and is one of the leading causes of death due to cancer invasion, metastasis, and resistance to therapies. Among its variants, triple-negative breast cancer (TNBC) is considered the most aggressive due to its early invasive and metastatic properties with poor prognosis. Kindlin-2, which is encoded by Fermitin family homolog 2 gene (FERMT2) has been associated with pathogenesis of several types of cancers of epithelial origin. Our previous studies have addressed the role of Kindlin-2 as a major regulator of the invasion-metastasis cascade in breast cancer by controlling several hallmarks of cancer in tumor cells. The contribution of mammary epithelial cell Kindlin-2 in the mammary glands to the process of tumor progression and metastasis has, however, not been investigated. Accordingly, we generated a floxed mouse strain by targeting the FREMT2 (K2lox/lox) locus using a CRISPR/Cas9-based editing strategy, followed by tissue-specific deletion of the Kindlin-2 in the basal subtype of the mammary epithelial cells (MECs) in the mouse mammary glands by crossing the K2lox/lox mice with K14-cre mice. Loss of Kindlin-2 in the basal MECs had no deleterious effects on mammary glands development, mouse development and fertility and lactation in mice bearing the Kindlin 2-deletetion phenotype and their progeny. However, in a syngeneic mouse model of BC, the loss of Kindlin-2 in MECs inhibited tumor growth and metastasis in mice inoculated with the aggressive murine TNBC E0771 cells when implanted directly in their mammary fat pads. Injecting the E0771 cells via the tail vein of Kindlin-2-deleted mice had, however, no effect on tumor colonization in the lungs, when compared to wild-type mice, clearly supporting a critical role of MECs Kindlin-2 in BC tumor growth and metastasis. Mechanistically, we found the MECs Kindlin-2-mediated inhibition of tumor growth and metastasis is through the regulation of the TGF-β/ERK MAP. kinase signaling axis, in a similar manner that our published studies showed that Kindlin-2 regulates this oncogenic pathway in the BC cells. Thus, our findings strongly suggest that Kindlin-2 supports BC oncogenesis in both the tumor cells and the MECs in the mammary glands, through the regulation of the TGF-β/EGF oncogenic signaling pathway. Therefore, therapeutic strategies targeting Kindlin-2 in both the cancer cells and the mammary glands may be necessary for a successful inhibition of BC tumors. Citation Format: Priyanka Rana, Wei Wang, Akram Alkrekchi, Katarzyna Bialkowska, Vesna Markovic, Edward F Plow, Elzbieta Pluskota, Khalid Sossey-Alaoui. Targeted deletion of Kindlin-2 in mouse mammary glands inhibits tumor growth, invasion and metastasis downstream of TGF-β/EGF oncogenic signaling pathway [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-06-02.