Stimulate Tumor Proliferation Research Articles

P17-4 Phenol stimulates tumor proliferation and metastasis through regulating β-3 adrenergic receptor

P17-4 Phenol stimulates tumor proliferation and metastasis through regulating β-3 adrenergic receptor

Identification of Hypoxia-Associated Signature in Colon Cancer to Assess Tumor Immune Microenvironment and Predict Prognosis Based on 14 Hypoxia-Associated Genes.

Colon cancer is the main malignant tumor of the digestive tract. Hypoxia is highly related to the occurrence, progression and tumor immune microenvironment (TIME) of cancer. The aim of this study was to identify a hypoxia-associated signature with high accuracy for predicting the prognosis and TIME of colon cancer. Download colon cancer data from the GEO and TCGA databases. A novel hypoxia risk model was identified to predict the prognosis of colon cancer patients. Subsequently, GSEA, TIME and mutation analysis were performed in the hypoxia high and low risk score groups. Finally, the signature gene ANKZF1 was selected for functional verification at the cellular level. A novel hypoxia risk model was identified. The risk score was significantly associated with poorer overall survival in colon cancer, and could be used as an independent prognostic factor for colon cancer. GSEA analysis found that the processes related to stimulate tumor proliferation and anti-apoptosis were significantly enriched in the hypoxia high risk score group. The expression of immunosuppressive cells and most immune checkpoints in the high risk score group was significantly higher than that in the low risk score group. In vitro cell experiments showed that knockdown the expression of ANKZF1 could inhibit the proliferation, migration and invasion of colon cancer cells. Hypoxia plays an important role in evaluating the TIME and predicting the prognosis of colon cancer.

Theranostic cobalt-55/58m for neurotensin receptor-mediated radiotherapy in vivo: A pilot study with dosimetry

Neurotensin receptor 1 (NTSR1) can stimulate tumor proliferation through neurotensin (NTS) activation and are overexpressed by a variety of cancers. The high binding affinity of NTS/NTSR1 makes radiolabeled NTS derivatives interesting for cancer diagnosis and staging. Internalization of NTS/NTSR1 also suggests therapeutic application with high LET alpha particles and low energy electrons. We investigated the therapeutic efficacy of [58mCo]Co-NOTA-NT-20.3 in vivo using murine models xenografted with NTSR1-positive HT29 human colorectal adenocarcinoma cells, and utilized [55Co]Co-NOTA-NT-20.3 for dosimetry. MethodsTargeting properties and cytotoxicity of [55/58mCo]Co-NOTA-NT-20.3 were assessed with HT29 cells. Female nude mice were xenografted with HT29 tumors and administered [55Co or 58mCo]Co-NOTA-NT-20.3 to evaluate pharmacokinetics or for therapy, respectively. Dosimetry calculations followed the Medical Internal Radiation Dose (MIRD) formalism and human absorbed dose rate per unit activity were obtained from OpenDose. The pilot therapy study consisted of two groups (each N = 3) receiving 110 ± 15 MBq and 26 ± 6 MBq [58mCo]Co-NOTA-NT-20.3 one week after tumor inoculation, and control (N = 3). Tumor sizes and masses were measured twice a week after therapy. Complete blood count and kidney histology were also performed to assess toxicity. ResultsHPLC measured radiochemical purity of [55,58mCo]Co-NOTA-NT-20.3 > 99 %. Labeled compounds retained NTS targeting properties. [58mCo]Co-NOTA-NT-20.3 exhibited cytotoxicity for HT29 cells and was >15× more potent than [58mCo]CoCl2. Xenografted tumors responded modestly to administered doses, but mice showed no signs of radiotoxicity. Absorbed dose to tumor and kidney with 110 MBq [58mCo]Co-NOTA-NT-20.3 were 0.6 Gy and 0.8 Gy, respectively, and other organs received less than half of the absorbed dose to tumor. Off-target radiation dose from cobalt-58g was small but reduces the therapeutic window. ConclusionThe enhanced in vitro cytotoxicity and high tumor-to-background led us to investigate the therapeutic efficacy of [58mCo]Co-NOTA-NT-20.3 in vivo. Although we were unable to induce tumor response commensurate with [177Lu]Lu-NT127 (NLys-Lys-Pro-Tyr-Tle-Leu) studies involving similar time-integrated activity, the absence of observed toxicity may constitute an opportunity for targeting vectors with improved uptake and/or retention to avoid the aftereffects of other high-LET radioactive emissions. Future studies with higher uptake, activity and/or multiple dosing regimens are warranted. The theranostic approach employed in this work was crucial for dosimetry analysis.

Anti-Hypoxia Nanoplatforms for Enhanced Photosensitizer Uptake and Photodynamic Therapy Effects in Cancer Cells.

Photodynamic therapy (PDT) holds great promise in cancer eradication due to its target selectivity, non-invasiveness, and low systemic toxicity. However, due to the hypoxic nature of many native tumors, PDT is frequently limited in its therapeutic effect. Additionally, oxygen consumption during PDT may exacerbate the tumor's hypoxic condition, which stimulates tumor proliferation, metastasis, and invasion, resulting in poor treatment outcomes. Therefore, various strategies have been developed to combat hypoxia in PDT, such as oxygen carriers, reactive oxygen supplements, and the modulation of tumor microenvironments. However, most PDT-related studies are still conducted on two-dimensional (2D) cell cultures, which fail to accurately reflect tissue complexity. Thus, three-dimensional (3D) cell cultures are ideal models for drug screening, disease simulation and targeted cancer therapy, since they accurately replicate the tumor tissue architecture and microenvironment. This review summarizes recent advances in the development of strategies to overcome tumor hypoxia for enhanced PDT efficiency, with a particular focus on nanoparticle-based photosensitizer (PS) delivery systems, as well as the advantages of 3D cell cultures.

Survival Benefit of Statin with Anti-Angiogenesis Efficacy in Lung Cancer-Associated Pleural Fluid through FXR Modulation.

Simple SummaryOur previous works showed that pleural fluid from lung cancer significantly induced endothelial proliferation, migration, and angiogenesis. Since endothelial metabolism was a key step in angiogenesis, we investigated the role of bile acid signaling and FXR expression in pleural angiogenesis. Elevated bile acid levels in lung-cancer-associated pleural fluid (LCPF) were characterized with positive FXR staining in pleural microvessels. We then confirmed the inhibitory effect of an FXR antagonist on LCPF-induced endothelial migration and angiogenesis. Due to the elevated protein expression in the cholesterol metabolism caused by LCPF, lipid-lowering agents with the efficacy needed to counteract LCPF-regulated angiogenesis were evaluated. Statin showed the potent efficacy needed to suppress LCPF-induced endothelial proliferation, migration, and angiogenesis through FXR inhibition. Following that, Kaplan–Meier analysis showed the survival benefit of statin exposure in patients with lung adenocarcinoma with LCPF. Our results suggest that targeting endothelial FXR signaling with statin treatment could ameliorate the angiogenesis activity of LCPF.Lung cancer-related pleural fluid (LCPF) presents as a common complication with limited treatment. Beyond its function in lipid digestion, bile acid was identified as a potent carcinogen to stimulate tumor proliferation. Previous research indicated a correlation between serum bile acid levels and the risk of developing several gastrointestinal cancers. Our study identified elevated bile acid levels in LCPF and increased farnesoid X receptor (FXR) expression as bile acid nuclear receptors in pleural microvessels of lung adenocarcinoma. Additionally, LCPF stimulated the expression of proteins involved in bile acid synthesis and cholesterol metabolism in HUVECs including CYP7A1, StAR, HMGCR, and SREBP2. LCPF-induced endothelial motility and angiogenesis were counteracted by using β-muricholic acid as an FXR antagonist. Moreover, we investigated the efficacy of cholesterol-lowering medications, such as cholestyramine, fenofibrate, and atorvastatin, in regulating LCPF-regulated angiogenesis. Along with suppressing endothelial proliferation and angiogenesis, atorvastatin treatment reversed cholesterol accumulation and endothelial junction disruption caused by LCPF. Statin treatment inhibited LCPF-induced endothelial FXR expression as well as the downstream proteins RXR and SHP. Based on the positive findings of suppressing endothelial angiogenesis, our group further incorporated the effect of statin on clinical patients complicated with LCPF. A Kaplan–Meier analysis revealed the clinical benefit of statin exposure in patients with lung adenocarcinoma with LCPF. Conclusively, our study demonstrated the ability of statin to alleviate LCPF-induced angiogenesis in patients with LCPF via FXR modulation.

Abstract P1-14-05: The impact of erythropoietin administration concomitantly with adjuvant anti-HER2 treatment on the patients’ outcome: Sub-analysis of the ALTTO study

Abstract Background: The erythropoietin (EPO) receptor activation can stimulate tumor proliferation and may be implicated in resistance to anti-HER2 therapies. EPO administration can be used for the management of chemotherapy-induced anemia. We aim to assess if EPO intake during the adjuvant anti-HER2 treatment impacts the outcomes of HER2-positive early breast cancer (EBC) patients. Methods: For this post hoc analysis of the ALTTO trial (NCT00490139), we categorized two cohorts according to the use of EPO during adjuvant anti-HER2 treatment: i) at least one EPO dose (EPO-cohort) vs ii) no EPO (non-EPO-cohort). We compared their baseline characteristics to identify potential confounders. The primary endpoint disease-free-survival (DFS), and the secondary endpoint overall survival (OS) were compared between these two cohorts, with subgroup analysis according to menopausal status, tumor characteristics, and anti-HER2 treatment. Kaplan Meier method, log-rank test, and Cox regression model adjusted for confounders were performed for DFS and OS (removal criterion p<0.10). The frequency of cardiovascular and thromboembolic adverse events (AEs) of EPO interest was compared between both cohorts. Results: Out of the 8,381 patients recruited in the ALTTO trial, 123 (1.5%) received EPO concomitantly with adjuvant treatment, 59 in the single HER2 blockade (Lapatinib (L)-alone or Trastuzumab (T)-alone) and 64 in dual blockade (T→L or T+L) arms. The median age of EPO-cohort patients was 54 (range: 27-74) years, and 39% were premenopausal, while most had pathologic tumor size >2 cm (56.9%), positive nodal status (58.5%), positive hormone receptor (HR) status (61.8%), and poorly differentiated or undifferentiated histologic grade (64.2%). Baseline characteristics were similar in both cohorts, except for the timing of chemotherapy, for which 93.5% of patients from the EPO-cohort were enrolled in the concurrent designs (2/2B) vs 44.2% in the non-EPO-cohort (p<0.001). Median FU was 6.9 years, with 6,409 (76.5%) patients still on follow-up. For the entire ALTTO cohort, the estimated 3- and 7-year DFS were 88.3% (95% CI: 87.6% - 89.0%) and 80.1% (95% CI: 79.1% - 81.0%), respectively. No difference in DFS by EPO administration (Yes/No) was observed (log-rank p=0.70). The effect of EPO remained non-significant when controlled for the four stratification factors and all other characteristics (p=0.91). The effect of EPO administration on DFS was significant for the subgroup of premenopausal women (p=0.041), favoring the EPO-cohort, noting that this result is based on small event numbers. The interaction was found significant in Cox analysis (p=0.024), remaining significant after adjustment for important prognostic variables (p=0.032). For the entire ALTTO cohort, 758 (9.0%) deaths occurred, with 3-year OS estimate 95.7% (95% CI: 95.2% - 96.1%) and 7-year OS estimate 89.9% (89.2% - 90.6%). The OS results are consistent with those for DFS regarding the lack of a significant association with EPO and the significance (p<0.10) of menopausal status and EPO interaction. In the EPO-cohort (n=123), eight (6.5%) AEs of EPO interest were recorded, while from the non-EPO-cohort (n=8,147), 417 (5.1%) reported similar AEs. Three fatal and 25 life-threatening AEs of EPO interest were recorded in the non-EPO-cohort, and none in the EPO-cohort. Conclusion: EPO administration to patients receiving adjuvant anti-HER2 treatment for HER2-positive EBC was safe and not associated with a harmful impact on survival outcomes, however the sample size of patients who received EPO is rather small. These findings contrast with data suggesting poorer outcomes with EPO, therefore further investigation of tumour microenvironment is needed to better understand these results, particularly in the premenopausal subgroup. Citation Format: Diogo Martins-Branco, Marie Kassapian, Véronique Debien, Rafael Caparica, Daniel Eiger, Urania Dafni, Charitini Andriakopoulou, Sarra El-Abed, Miguel Izquierdo, Malou Vicente, Saranya Chumsri, Martine Piccart-Gebhart, Alvaro Moreno-Aspitia, Ann Søegaard Knop, Janine Lombard, Evandro de Azambuja. The impact of erythropoietin administration concomitantly with adjuvant anti-HER2 treatment on the patients’ outcome: Sub-analysis of the ALTTO study [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 P1-14-05.

The Relationship of Fibroblast Activation Protein (FAP) Expression to Clinicopathologic Factors of Papillary Thyroid Carcinoma

BackgroundPapillary thyroid carcinoma is the most common subtype among thyroid cancers. The interaction between cancer cells and tumormicroenvironment plays roles in cancer proliferation and invasiveness. One of important components of tumor microenvironmentthat could stimulate tumor proliferation and invasiveness is activated fibroblast, known as carcinoma associated fibroblasts (CAFs).The aim of this research is to analysed FAP expression is associated with clinicopathologic characteristics.MethodsThis retrospective descriptive observational study with serial cases design was conducted in 40 samples of the paraffin blocks oftissues registered in Department of Anantomical Pathology Faculty of Medicine University of Sriwijaya/Dr. Moh. Hoesin PalembangHospital from January 1st, 2017 to October 31st, 2019. All samples were immunostained with FAP antibody. The expressions of FAPwere evaluated in accordance with the results of the previous study. Statistical test was performed by using Chi-square andSpearman-rho statistical test. Statistical analysis was performed by using SPSS version 23.0.ResultsOur data indicated that patients who were ≥45 years old (21 cases; 52.5%), women (31 cases; 77.5%) suffered from this cancer themost. The stromal type found in ten classic, follicular, solid and columnar variance were pauci (25 cases; 62.6%), desmoplastic (8cases;20%), sclerosis (5 cases; 12.5%) and inflammatory (2 cases; 5%). The expression of FAP in tumor stroma has a significantcorrelation with histopathology subtypes (p=0.028) and stromal type (p=0.000).ConclusionFAP was expressed in tumor epithelial cells and fibroblast stromal tumour of papillary thyroid carcinoma. FAP expression in stromaltumor between histopathologic subtype and stromal type showed significant correlation

Newly-Discovered Neural Features Expand the Pathobiological Knowledge of Blastic Plasmacytoid Dendritic Cell Neoplasm.

Simple SummaryFor the first time, neuronal features are described in blastic plasmacytoid dendritic cell neoplasm (BPDCN) by a complex array of molecular techniques, including microRNA and gene expression profiling, RNA and Chromatin immunoprecipitation sequencing, and immunohistochemistry. The discovery of unexpected neural features in BPDCN may change our vision of this disease, leading to the designing of a new BPDCN cell model and to re-thinking the relations occurring between BPDCN and nervous system. The observed findings contribute to explaining the extreme tumor aggressiveness and also to propose novel therapeutic targets. In view of this, the identification, in this work of new potential neural metastatic inducers might open the way to therapeutic approaches for BPDCN patients based on the use of anti-neurogenic agents.Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive hematologic malignancy originating from plasmacytoid dendritic cells (pDCs). The microRNA expression profile of BPDCN was compared to that of normal pDCs and the impact of miRNA dysregulation on the BPDCN transcriptional program was assessed. MiRNA and gene expression profiling data were integrated to obtain the BPDCN miRNA-regulatory network. The biological process mainly dysregulated by this network was predicted to be neurogenesis, a phenomenon raising growing interest in solid tumors. Neurogenesis was explored in BPDCN by querying different molecular sources (RNA sequencing, Chromatin immunoprecipitation-sequencing, and immunohistochemistry). It was shown that BPDCN cells upregulated neural mitogen genes possibly critical for tumor dissemination, expressed neuronal progenitor markers involved in cell migration, exchanged acetylcholine neurotransmitter, and overexpressed multiple neural receptors that may stimulate tumor proliferation, migration and cross-talk with the nervous system. Most neural genes upregulated in BPDCN are currently investigated as therapeutic targets.

Analyses of Fibroblast Activation Protein (FAP) Expression Rate to Clincopathology Factors and Chemotherapy Response in Ovarian Epithelial Malignant Tumors

BackgroundEpithelial ovarian carcinoma is the most common cause of death among gynecological malignancies in the women. The interactionamong cancer cells and tumor microenvironment plays roles in cancer proliferation and invasiveness. One of important component oftumor microenvironment that could stimulate tumor proliferation and invasiveness, is fibroblast. Activated fibroblasts also known ascarcinoma associated fibroblasts (CAFs). The purpose of research is to analyzed FAP expressions to clinicopathologic characteristic(histopathology subtype and clinical stadium of FIGO) and chemotherapeutic responses.MethodsThis study is a retrospective observational analytic study with cross-sectional designs was undertaken during January 1, 2016 to June30, 2018. The sample was 55 paraffin block sample epithelial ovarian carcinomas consists of 11 samples for each histopathologysubtype collected used purposive sampling technique. All samples were immunostained with FAP antibody. FAP expressions wasevaluated based on Min Shi et al studies. It analyzed stained area/ proportion (P) and intensity (I). Statistical analysis using chi-squarewas performed using SPSS version 17.ResultsPatients of 40-49 years old (24; 43.6%), clinical stadium is III (30; 54.5%), Complete response (CR) of chemotherapeutic response(38; 69.1%). The statistic analyses between histopathology subtypes (p=0.038), clinical stadium of FIGO (p=0.000) and chemotherapyresponse (p=0.000) showed significant differences.ConclusionFAP expressions could showed correlation between clinicopathologic characteristic (histopathology subtype and clinical stadium ofFIGO) and chemotherapeutic response.

Analyzes of Interleukin-17 Expression and Clinicopathological Characteristics of Breast Carcinoma

BackgroundInteraction between cancer cells and tumor microenvironment plays roles in cancer proliferation and invasiveness. Many type ofcytokine produced by inflammatory cells in tumor stroma could stimulate tumor proliferation and invasiveness, such as interleukin-17.The aim of this study is to determine the correlation between IL-17 expression and clinicopathological characteristics of breast cancer.MethodsSerial cases study was performed by enrolling 40 paraffin block of breast cancer patients. Clinicopathological characteirstics werenoted, including age, histopathological subtype and grade, TILs, lymphovascular invasion and molecular subtype. TILs were scoredby denomination area of stromal tissue occupied by mononuclear cells over total stromal area. Expression of IL-17 was examined byusing immunohistochemistry with anti-IL-17 antibody and counted from stromal mononuclear cells cytoplasm staining from five areas.The average value was used as a cut-off point in determining level of expression. Data were analysed by using SPSS v.22.ResultsMajority of the patients were more than 40 years old (75%), histopathological grade III (77.5%), without lymphovascular invasion(62.5%), luminal B molecular subtype (57.5%), and non-dominant TILs (95%). IL-17 expression was not significantly correlated withclinicopathological characteristics with p value=0.079, 0.600, 0.739, 0.883 and 1.000 respectively for age, histological grade,lymphovascular invasion, molecular subtype and TILs.ConclusionInterleukin-17 expression and clinicopathological on characteristics of breast carcinoma between histopathological grade, TILs, andlymphovascular invasion showed no significant differences. There was a tendency that the higher IL-17 expression, the higherhistopathological grade, the lower TILs and positive lymphovascular invasion

Abstract PO-034: Non-random spatial clustering of tumor associated macrophages is associated with poor survival in metastatic RCC patients receiving immunotherapy

Abstract INTRODUCTION Tumor associated macrophages (TAM) stimulate tumor proliferation and facilitate immune escape via production of immunosuppressive cytokines. We hypothesize that non-random spatial clustering of TAMs within the tumor is associated with poor survival in RCC patients. METHODS Primary and metastatic tumor specimens were obtained from 39 patients with metastatic RCC who received immunotherapy (IT). 29 patients (74.3%) also received targeted therapy (TT) at some point during their disease course. Sections from the tumor compartment underwent immunohistochemical (IHC) staining for pan-macrophage marker CD68, and M2 markers CD163 and CD206. Digital pathologic analysis was used to convert the digital images to spatial point pattern plots (PPP). A probability density function (PDF) for the distance between cells was calculated under the assumption that cells can be located anywhere in the slide with equal probability. The PDF was then compared to empirical histograms of cells derived from the PPP. Deviations from the PDF, the expected random distribution, reveal evidence of cell clustering or dispersion. We assessed deviations with the Kolmogorov-Smirnov (KS) test. Patients were stratified by the median value into high and low KS groups, and survival outcomes were compared between the two groups using a Cox regression. A high KS statistic indicates a good fit between the two curves (in this case, randomness), and a low KS statistic indicates a poor fit (in this case, dispersion or clustering). RESULTS Among primary tumor specimens, spatial clustering of CD68+ cells was associated with worse overall survival (OS) (HR -1.7, p=0.015), worse OS from IT initiation (HR -1.5, p=0.025), and no difference in OS from TT initiation (HR -0.4, p=0.48). Among metastatic tumor specimens, spatial clustering of CD68+ cells was not associated with any of the three survival outcomes (OS: HR 0.9, p=0.11; OS from IT: HR 0.7, p=0.20; OS from TT: HR 0.0, p=0.97). Spatial clustering of CD163+ cells was not associated with any of the three survival outcomes for either primary and metastatic sites. Spatial clustering of CD206+ cells was not associated with any of the three survival outcomes for either primary and metastatic sites. CONCLUSION Non-random spatial clustering of CD68+ TAMs in the primary tumor specimen was associated with worse survival, driven primarily by worse survival from initiation of immunotherapy. These findings corroborate prior reports of TAMs eliciting an immunosuppressive effect on the tumor-immune microenvironment, introduce novel methodology for quantifying spatial heterogeneity, and demonstrate the novel finding of a clinically significant effect of TAM spatial clustering on overall survival. Citation Format: Gregory J. Kimmel, Nicholas H. Chakiryan, Youngchul Kim, Ali J. Hajiran, Ahmet M. Aydin, Jonathan V. Nguyen, Carlos M. Moran-Segura, Jasreman Dhillon, Philipp Altrock, Brandon J. Manley. Non-random spatial clustering of tumor associated macrophages is associated with poor survival in metastatic RCC patients receiving immunotherapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-034.

Abstract A30: Novel T-cell engaging antibodies against L1CAM for neuroblastoma

Abstract Background: T cell-mediated immunotherapy has shown great promise in the treatment of human cancers. Bispecific antibodies (BsAbs) that engage CD3 on T cells and a tumor antigen on target cells can induce polyclonal T cell-mediated cytotoxicity against both leukemia (expressing CD19) and solid tumors (expressing HER-2 or GD2), unrestricted by MHC. The L1 cell adhesion molecule (L1CAM) is a glycoprotein consisting of 6 Ig-like domains and 5 fibronectin-like repeats in the ectodomain. In normal tissues, L1CAM expression is restricted to neurons, renal tubules, and skin at low levels and plays an important role in brain development, including neuronal migration and differentiation. Interestingly, L1CAM is also overexpressed on various pediatric cancers including neuroblastoma (NB), and stimulates tumor proliferation, migration, and metastasis. Chimeric IgG1 antibody, chCE7, targeting the 6th Ig-like domain of human L1CAM, had high affinity (KD = 0.63 nM) and when 131I-labeled, had superior sensitivity and specificity over 131I-MIBG in patients with metastatic NB. However, chCE7 lacked ADCC ability in preclinical studies and no major clinical responses have been reported for anti-L1CAM chimeric antigen receptor (CAR) modified T cells (NCT02311621). Methods: By CDR grafting, the murine antibody E71 (specific for 2nd Ig-like domain with KD = 2.23 nM) was humanized to huE71, and huE71-BsAb using the IgG(L)-scFv BsAb platform previously described (Xu et al., Can Immunol Res 2016) was generated and compared to huE72 (the humanized version of CE7)-BsAb. The BsAbs were produced in CHO-S cells and purified by protein A affinity chromatography. Size homogeneity was confirmed by SEC HPLC. Binding to L1CAM and CD3, or to tumors and T cells was measured by SPR (Biacore) or by FACS, respectively. In vitro T-cell activation and cytokine release were assessed by FACS and by ELISA, respectively. In vitro tumor cytotoxicity was measured by 4-hour 51Cr release at an E:T ratio of 10:1. In vivo antitumor effect was assessed in Balb/c-Rag2-/-IL-2R-c-KO (DKO) mice xenografted with subcutaneous (s.c.) NB cell lines or patient-derived xenografts (PDX). Tumor targeting was performed by using 89Zr-labeled huE71 and huE72 IgG1 in xenografted nude mice. Results: Both BsAbs showed a molecular size of 210kDa with >90% purity. When assessed by SPR for binding to the L1CAM, huE71-BsAb retained its parental affinity, while that of huE72-BsAb decreased by 8-fold (KD of 5.15nM and 5.28nM, respectively). As expected from the IgG(L)-scFv design, both BsAbs showed lower binding to CD3(+) T cells when compared with huOKT3 IgG1. Both BsAbs induced CD25 expression on CD4(+) or CD8(+) T cells after 96 hours of culture in the presence of L1CAM(+) IMR32 NB cell line, and CD8(+) T cells showed more activation in the presence of huE72-BsAb. Both BsAbs induced similar level of Th1 cytokine (e.g. TNFα) from PBMCs following 24 hours of culture in the presence of IMR32. Both BsAbs mediated T-cell cytotoxicity on L1CAM(+) cell lines with pM EC50s. In nude mice xenografted with s.c. L1CAM(+) cancer cell line, intravenous (i.v.) huE71 IgG1 showed higher tumor targeting efficiency than huE72 IgG1. In DKO mice xenografted with s.c. IMR32 (carrying luciferase reporter gene) and s.c. PBMCs, i.v. huE71-BsAb significantly suppressed tumor growth while huE72-BsAb had no antitumor effect. Similar results were observed in an s.c. L1CAM(+) NB PDX model when PBMCs were injected i.v. Conclusions: Despite near-identical in vitro binding characteristics, BsAbs targeting different epitopes on the same target have strikingly different antitumor properties in vivo. HuE71-BsAb is a potential T cell engaging immunotherapeutic for NB and other L1CAM(+) solid tumors. Citation Format: Maya Suzuki, Hong Xu, Hongfen Guo, Brandon Nemieboka, Zhihao Wu, Jason Lewis, Nai-Kong V. Cheung. Novel T-cell engaging antibodies against L1CAM for neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A30.

Adenosine can thwart antitumor immune responses elicited by radiotherapy : Therapeutic strategies alleviating protumor ADO activities.

By studying the bioenergetic status we could show that the development of tumor hypoxia is accompanied, apart from myriad other biologically relevant effects, by a substantial accumulation of adenosine (ADO). ADO has been shown to act as a strong immunosuppressive agent in tumors by modulating the innate and adaptive immune system. In contrast to ADO, standard radiotherapy (RT) can either stimulate or abrogate antitumor immune responses. Herein, we present ADO-mediated mechanisms that may thwart antitumor immune responses elicited by RT. An overview of the generation, accumulation, and ADO-related multifaceted inhibition of immune functions, contrasted with the antitumor immune effects of RT, is provided. Upon hypoxic stress, cancer cells release ATP into the extracellular space where nucleotides are converted into ADO by hypoxia-sensitive, membrane-bound ectoenzymes (CD39/CD73). ADO actions are mediated upon binding to surface receptors, mainly A2A receptors on tumor and immune cells. Receptor activation leads to a broad spectrum of strong immunosuppressive properties facilitating tumor escape from immune control. Mechanisms include (1) impaired activity of CD4 (+) T and CD8 (+) T, NK cells and dendritic cells (DC), decreased production of immuno-stimulatory lymphokines, and (2) activation of Treg cells, expansion of MDSCs, promotion of M2 macrophages, and increased activity of major immunosuppressive cytokines. In addition, ADO can directly stimulate tumor proliferation and angiogenesis. ADO mechanisms described can thwart antitumor immune responses elicited by RT. Therapeutic strategies alleviating tumor-promoting activities of ADO include respiratory hyperoxia or mild hyperthermia, inhibition of CD39/CD73 ectoenzymes or blockade of A2A receptors, and inhibition of ATP-release channels or ADO transporters.

Acid gradient across plasma membrane can drive phosphate bond synthesis in cancer cells: acidic tumor milieu as a potential energy source.

Aggressive cancers exhibit an efficient conversion of high amounts of glucose to lactate accompanied by acid secretion, a phenomenon popularly known as the Warburg effect. The acidic microenvironment and the alkaline cytosol create a proton-gradient (acid gradient) across the plasma membrane that represents proton-motive energy. Increasing experimental data from physiological relevant models suggest that acid gradient stimulates tumor proliferation, and can also support its energy needs. However, direct biochemical evidence linking extracellular acid gradient to generation of intracellular ATP are missing. In this work, we demonstrate that cancer cells can synthesize significant amounts of phosphate-bonds from phosphate in response to acid gradient across plasma membrane. The noted phenomenon exists in absence of glycolysis and mitochondrial ATP synthesis, and is unique to cancer. Biochemical assays using viable cancer cells, and purified plasma membrane vesicles utilizing radioactive phosphate, confirmed phosphate-bond synthesis from free phosphate (Pi), and also localization of this activity to the plasma membrane. In addition to ATP, predominant formation of pyrophosphate (PPi) from Pi was also observed when plasma membrane vesicles from cancer cells were subjected to trans-membrane acid gradient. Cancer cytosols were found capable of converting PPi to ATP, and also stimulate ATP synthesis from Pi from the vesicles. Acid gradient created through glucose metabolism by cancer cells, as observed in tumors, also proved critical for phosphate-bond synthesis. In brief, these observations reveal a role of acidic tumor milieu as a potential energy source and may offer a novel therapeutic target.

Functional EpoR pathway utilization is not detected in primary tumor cells isolated from human breast, non-small cell lung, colorectal, and ovarian tumor tissues.

Several clinical trials in oncology have reported increased mortality or disease progression associated with erythropoiesis-stimulating agents. One hypothesis proposes that erythropoiesis-stimulating agents directly stimulate tumor proliferation and/or survival through cell-surface receptors. To test this hypothesis and examine if human tumors utilize the erythropoietin receptor pathway, the response of tumor cells to human recombinant erythropoietin was investigated in disaggregated tumor cells obtained from 186 patients with colorectal, breast, lung, ovarian, head and neck, and other tumors. A cocktail of well characterized tumor growth factors (EGF, HGF, and IGF-1) were analyzed in parallel as a positive control to determine whether freshly-isolated tumor cells were able to respond to growth factor activation ex vivo. Exposing tumor cells to the growth factor cocktail resulted in stimulation of survival and proliferation pathways as measured by an increase in phosphorylation of the downstream signaling proteins AKT and ERK. In contrast, no activation by human recombinant erythropoietin was observed in isolated tumor cells. Though tumor samples exhibited a broad range of cell-surface expression of EGFR, c-Met, and IGF-1R, no cell-surface erythropoietin receptor was detected in tumor cells from the 186 tumors examined (by flow cytometry or Western blot). Erythropoiesis-stimulating agents did not act directly upon isolated tumor cells to stimulate pathways known to promote proliferation or survival of human tumor cells isolated from primary and metastatic tumor tissues.

Increased extracellular pressure stimulates tumor proliferation by a mechanosensitive calcium channel and PKC-β

Large tumors exhibit high interstitial pressure heightened by growth against the constraining stroma. Such pressures could stimulate tumor proliferation via a mechanosensitive ion channel. We studied the effects of 0–80 mmHg increased extracellular pressure for 24 h on proliferation of SW620, Caco-2, and CT-26 colon; MCF-7 breast; and MLL and PC3 prostate cancer cells, and delineated its mechanism in SW620 cells with specific inhibitors and siRNA. Finally, we compared NF-kB, phospho-IkB and cyclin D1 immunoreactivity in the high pressure centers and low pressure peripheries of human tumors. Pressure-stimulated proliferation in all cells. Pressure-driven SW620 proliferation required calcium influx via the T-type Ca2+ channel Cav3.3, which stimulated PKC-β to invoke the IKK-IkB–NF–kB pathway to increase proliferation and S-phase fraction. The mitotic index and immunoreactivity of NF-kB, phospho-IkB, and cyclin D1 in the center of 28 large human colon, lung, and head and neck tumors exceeded that in tumor peripheries. Extracellular pressure increases [Ca2+]i via Cav3.3, driving a PKC-β- IKK- IkB–NF–kB pathway that stimulates cancer cell proliferation. Rapid proliferation in large stiff tumors may increase intratumoral pressure, activating this pathway to stimulate further proliferation in a feedback cycle that potentiates tumor growth. Targeting this pathway may inhibit proliferation in large unresectable tumors.

TGF-beta Signaling in Cancer Treatment

The transforming growth factor-beta (TGF-β ) belongs to a superfamily of cytokines that act on protein kinase receptors at the plasma membrane to induce a plethora of biological signals that regulate cell growth and death, differentiation, immune response, angiogenesis and inflammation. Dysregulation of its pathway contributes to a broad variety of pathologies, including cancer. TGF-β is an important regulatory tumor suppressor factor in epithelial cells, where it early inhibits proliferation and induces apoptosis. However, tumor cells develop mechanisms to overcome the TGF-β -induced suppressor effects. Once this occurs, cells may respond to this cytokine inducing other effects that contribute to tumor progression. Indeed, TGF-β induces epithelial-mesenchymal transition (EMT), a process that is favored in tumor cells and facilitates migration and invasion. Furthermore, TGF-β mediates production of mitogenic growth factors, which stimulate tumor proliferation and survival. Finally, TGF-β is a well known immunosuppressor and pro-angiogenic factor. Many studies have identified the overexpression of TGF-β 1 in various types of human cancer, which correlates with tumor progression, metastasis, angiogenesis and poor prognostic outcome. For these reasons, different strategies to block TGF-β pathway in cancer have been developed and they can be classified in: (1) blocking antibodies and ligand traps; (2) antisense oligos; (3) TβRII and/or ALK5 inhibitors; (4) immune response-based strategies; (5) other inhibitors of the TGF-β pathway. In this review we will overview the two faces of TGF-β signaling in the regulation of tumorigenesis and we will dissect how targeting the TGF-β pathway may contribute to fight against cancer.

Adipose tissue and cancer: a high risk tandem

Adipose tissue is found in close proximity whith many invasive cancers. In breast cancer, early local tumour invasion results in close interactions of cancer cells with fully differentiated adipocytes. Aside from their energy-storing function, mature adipocytes are also active endocrine cells prone to influence tumour behaviour through heterotypic signaling processes. After a short description of anatomical depots specificities of adipose tissue, we describe the phenotypic changes induced by tumor secretion in tumour-surrounding adipocytes. These cells (that we named CAA for cancer-associated adipocytes) by their ability to secrete pro-inflammatory cytokines, extra-cellular matrix proteins and proteases involved in its remodeling, as well as to release free fatty acid, stimulate tumor proliferation, invasiveness and drug resistance. These results support the concept that adipocytes participate in a deleterious crosstalk with cancer cells to support tumour progression, that might be amplified in obesity conditions and explain the poor prognosis of cancers observed in this subset of patients.

Abstract 392: The role of post surgical inflammation in tumor progression in breast cancer

Abstract Introduction The process of chronic inflammation inducing malignant transformation is well known and recognized in several disease states, including ulcerative colitis (colorectal carcinoma), chronic wounds (squamous cell carcinoma), and hepatitis B infection (hepatocellular carcinoma) as common examples. Recent evidence suggests that this process may be relevant in other tumor types and may be influenced by inflammation arising secondary to interventional procedures. The purpose of this study is to investigate the potential role of inflammatory mediators resulting from the post-surgical healing process on tumor proliferation and progression to metastasis in breast cancer. Methods We used MMTV-PyV MT female mice (transgenic mice expressing the middle T antigen of the polyomavirus under the control of the MMTV promoter/enhancer) as a mouse model of human epidermal breast cancer. Mice underwent a surgical punch biopsy (2 mm) in the center of the tumor, or underwent the same procedure (transient anesthesia) without biopsy. Mice were paired according to the tumor size at the time of biopsy. Mice were then euthanized 8 days after biopsy, and lungs were harvested and histologically examined for the presence of micrometastases. To further evaluate the effect of inflammation in the development of metastases, a separate subset of mice were treated with ibuprofen versus vehicle (placebo control) for three days after biopsy. Results No substantial difference in the primary tumor growth was observed after biopsy between the various groups. However, a greater number of metastases were found in the lungs of mice that underwent biopsy relative to the number in mice that did not receive biopsy. The number of metastases was reduced in the set of mice receiving ibuprofen treatment following the biopsy procedure (9.3 vs. 25 p<0.05). Conclusion Our study provides evidence to suggest that inflammatory mediators arising secondary to the healing process may stimulate tumor proliferation and metastasis. These findings may hold significance for the timing of breast cancer treatment following diagnostic needle biopsy, as well as for the potential impact of excisional biopsy on the outcome of patients with breast cancer. Further research into the mechanisms of this interaction may help to increase our understanding of breast cancer biology and may yield new strategies (e.g. anti-inflammatory medication) to optimize treatment and outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 392. doi:1538-7445.AM2012-392

Epor Is Not Expressed or Functional in Primary Tumor Cells Isolated Directly From Human Primary and Metastatic Epithelial Tumors Including Breast, Non-Small Cell Lung, Colorectal, and Ovarian Tissues

Abstract 4781Several clinical trials have reported increased mortality or disease progression associated with erythropoiesis-stimulating agents (ESA) in patients with cancer. One hypothesis proposes that ESAs stimulate tumor proliferation and or survival directly through binding and activating cell-surface receptors (EpoR). Expression and function of cell surface EpoR in human tumor tissue is controversial and has not been extensively investigated using validated reagents. MethodsEpoR expression and function was analysed in disaggregated primary tumor cells obtained from surgically resected human tumor tissues from 186 patients: colorectal (n=46), breast (n=34, including 8 Her2+ tumors defined by HercepTest™), non small cell lung (n=41), ovarian (n=35), Head and Neck (n=5) and other tumor types (n=8). Metastatic tissues were also analysed: colon (n=2), ovary (n=3), lung (n=2), pancreatic (n=1) as well as tumors from pre-treated patients: colorectal (oxaliplatin, bevacizumab; n=1), breast (cyclophosphamide, methotrexate, fluorouracil; n=2), breast (radiation, docetaxel cyclophosphamide, Paclitaxel; n=1), non small cell lung (radiation; n=2), ovarian (carboplatin and Paclitaxel; n=2).To examine if cell surface EpoR is expressed in the tumor cell compartment, flow cytometry analysis was carried out using a validated, EpoR-specific antibody (mAb 307). To evaluate if tumor cells respond functionally to recombinant human Epo (rHuEpo), levels of phosphorylated downstream signaling proteins (Akt, Erk1/2 and STAT5) were analysed by intracellular flow cytometry using phospho-specific antibodies following ex vivo stimulation with a titration of rHuEpo (0U [vehicle] to 300U/mL for 5 and 30 min). Expression of known tumor growth factor receptors (IGF-1R, c-Met and EGFR) was analyzed as positive controls. Additionally, the response of Akt, Erk and STAT pathways to stimulation with a tumor growth factor cocktail (“GF”: EGF, HGF and IGF-1), was used as a control for pathway integrity. The sensitivity of all flow based assays was validated through the analysis of EpoR expression and function in erythroid precursor cells (EPCs) across a time course of differentiation from primary human bone marrow samples. ResultsThough tumor samples exhibited cell-surface expression of EGFR, c-Met, and IGF-1R, no cell-surface EpoR was detected in tumor cells from any of the 186 tumors examined by flow cytometry. Mean expression relative to negative control: EGFR = 2.80 (95% CI, 1.63–3.97), c-Met = 5.28 (95% CI, 4.39–6.17), IGF-1R = 8.03 (95% CI, 6.81–9.24) and EpoR (0.69 (95% CI, 0.64–0.73). No EpoR was detected by Western blot analysis of tumor lysates. As shown in Table 1, treatment with the GF cocktail stimulated phosphorylation of Akt and Erk in all cohorts. However, no activation of these pathways (or STAT5) by rHuEpo was observed in any of tumors even at high supra-pharmacological concentrations of rHuEpo (up to 300U/mL) and extended stimulation times (up to 30 mins).Table 1Mean response of signaling proteins to treatment with tumor growth factor cocktail (“GF”) and rHuEpo (300U/mL treatment shown). All values shown relative to vehicle controlPatient cohortpAktpErkGFrHuEpoGFrHuEpoBreast2.491.001.310.99Colon1.641.001.340.97Lung1.860.991.520.99Ovarian2.101.001.770.98Other2.071.021.340.94Pre-treated1.721.011.190.95Metastatic3.160.971.651.00Analysis of EpoR expression and function during differentiation in-vitro of EPCs revealed detectable EpoR cell surface expression (2.13-fold above baseline) and function (e.g. pSTAT5: 1.39-fold relative to vehicle at 300 U/mL rHuEpo) as early as day 1 when EPCs (CD36+/CD34-) comprised only 6.9% of the population. By day 8, (>90% EPCs) EpoR expression was detectable at 6.36-fold relative to baseline and pSTAT5 activation was observed 5.6-fold relative to vehicle at 300U/mL rHuEpo. ConclusionUsing sensitive methods no evidence of EpoR expression or function in primary human tumor cells isolated directly from primary and metastatic tumors was observed in multiple epithelial tumor types. Expression and functional activation of EGFR, c-Met and IGF1R was readily detectable. Therefore, this study suggests that it is unlikely that rHuEpo acts as a growth factor for primary human tumor cells. Disclosures:McCaffery:Amgen: Employment, Equity Ownership. Rossi:Amgen: Employment, Equity Ownership. Paweletz:Amgen: Employment, Equity Ownership. Tudor:Amgen: Employment, Equity Ownership. Elliott:Amgen Inc: Employment. Fitzpatrick:Amgen Inc.: Employment, Equity Ownership. Patterson:Amgen: Employment, Equity Ownership.