Ic Injection Research Articles

Abstract 2808: Characterization of the metastatic behavior and gene expression profile (RNAseq) of different melanoma cell lines: a comprehensive in vivo model

Abstract Background: Metastasis is the major cause of death in malignant melanoma. Several factors, including clinical-pathological and tumor biological features may restrain prognosis. Molecular mechanisms regulating melanoma progression and metastasis have been partially discovered, and thus we developed an in vivo model of metastatic melanoma to investigate potential genes implicated in these events. Materials and methods: To evaluate the in vivo metastatic activity of five different melanoma cell lines (LCP, LCM, WM266, SK-Mel28 and A375), we completed intra-cardiac (ic) injection of 1x106 luminescent cells or PBS as control in three NOD-SCID mice per cell line. After 3 weeks, mice were studies by in vivo bioluminescence imaging (IVIS Lumina LT) to measure the mean radiance (p/sec/cm2/sr), that we arbitrarily considered as a surrogate of the total metastatic tumor burden (t-MTB). Animals were euthanized at the humane endpoints achievement and underwent X-ray evaluation for bone metastasis detection. The Kaplan-Meyer curves described the time to sacrifice from ic injection. Mouse necropsy identified metastatic organs that were explanted and processed for both histology and gene expression analysis. Melanoma cell lines were profiled for gene expression (RNAseq) of 118 genes notably involved in cancer progression and metastasis. Quantitative RT-PCR (qRT-PCR) explored the expression of a restrict number of genes on formalin-fixed paraffin-embedded (FFPE) metastatic samples from euthanized animals. Results: All melanoma cell lines demonstrated a metastatic behavior following ic injection with a variable attitude to produce bone and visceral metastasis. Mice injected with LCP, LCM and WM266 showed a lower t-MTB and increased survival as compared to A375 and SK-Mel28. Thus, we arbitrarily defined LCP, LCM and WM-266 cell lines ‘poorly metastatic' (group A), while A375 and SK-Mel28 ‘highly metastatic' (group B). The principal component analysis and the unsupervised hierarchical clustering (118 gene transcriptome heatmap) revealed similar gene expression profiling among cell lines grouped for the metastatic attitude (Figure 3). The gene expression analysis (Figure 4) performed on FFPE samples identified five deregulated genes (WNT5A, COL6A3, PTHLH, SOX9 and SERPINE1) between A and B. Conclusions: We describe the metastatic capacity of five melanoma cell lines. Gene expression profiling revealed the activation of five genes as putatively responsible for the high aggressiveness of A375 and SK-Mel28 cells. These results suggest to investigate these genes in a clinical setting and their possible application as druggable target for future therapeutic strategies. Citation Format: Francesco Mannavola, Domenica Lovero, Claudia Felici, Luigia Stefania Stucci, Camillo Porta, Marco Tucci. Characterization of the metastatic behavior and gene expression profile (RNAseq) of different melanoma cell lines: a comprehensive in vivo model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2808.

Inter-Metastatic Heterogeneity of Tumor Marker Expression and Microenvironment Architecture in a Preclinical Cancer Model.

Background: Preclinical drug development studies rarely consider the impact of a candidate drug on established metastatic disease. This may explain why agents that are successful in subcutaneous and even orthotopic preclinical models often fail to demonstrate efficacy in clinical trials. It is reasonable to anticipate that sites of metastasis will be phenotypically unique, as each tumor will have evolved heterogeneously with respect to gene expression as well as the associated phenotypic outcome of that expression. The objective for the studies described here was to gain an understanding of the tumor heterogeneity that exists in established metastatic disease and use this information to define a preclinical model that is more predictive of treatment outcome when testing novel drug candidates clinically. Methods: Female NCr nude mice were inoculated with fluorescent (mKate), Her2/neu-positive human breast cancer cells (JIMT-mKate), either in the mammary fat pad (orthotopic; OT) to replicate a primary tumor, or directly into the left ventricle (intracardiac; IC), where cells eventually localize in multiple sites to create a model of established metastasis. Tumor development was monitored by in vivo fluorescence imaging (IVFI). Subsequently, animals were sacrificed, and tumor tissues were isolated and imaged ex vivo. Tumors within organ tissues were further analyzed via multiplex immunohistochemistry (mIHC) for Her2/neu expression, blood vessels (CD31), as well as a nuclear marker (Hoechst) and fluorescence (mKate) expressed by the tumor cells. Results: Following IC injection, JIMT-1mKate cells consistently formed tumors in the lung, liver, brain, kidney, ovaries, and adrenal glands. Disseminated tumors were highly variable when assessing vessel density (CD31) and tumor marker expression (mkate, Her2/neu). Interestingly, tumors which developed within an organ did not adopt a vessel microarchitecture that mimicked the organ where growth occurred, nor did the vessel microarchitecture appear comparable to the primary tumor. Rather, metastatic lesions showed considerable variability, suggesting that each secondary tumor is a distinct disease entity from a microenvironmental perspective. Conclusions: The data indicate that more phenotypic heterogeneity in the tumor microenvironment exists in models of metastatic disease than has been previously appreciated, and this heterogeneity may better reflect the metastatic cancer in patients typically enrolled in early-stage Phase I/II clinical trials. Similar to the suggestion of others in the past, the use of models of established metastasis preclinically should be required as part of the anticancer drug candidate development process, and this may be particularly important for targeted therapeutics and/or nanotherapeutics.

Altered circadian rhythms in a mouse model of neurodevelopmental disorders based on prenatal maternal immune activation

Individuals with neurodevelopmental disorders, such as schizophrenia and autism spectrum disorder, exhibit various sleep and circadian rhythm disturbances that often persist and worsen throughout the lifespan. To study the interaction between circadian rhythm disruption and neurodevelopmental disorders, we utilized a mouse model based on prenatal maternal immune activation (MIA). We hypothesized that MIA exposure would lead to impaired circadian locomotor activity rhythms in adult mouse offspring. We induced MIA by injecting pregnant dams with polyinosinic:polycytidylic acid (poly IC) at embryonic day 9.5, then aged resulting offspring to adulthood. We first confirmed that poly IC injection in pregnant dams elevated plasma levels of pro- and anti-inflammatory cytokines and chemokines. We then placed adult offspring in running wheels and subjected them to various lighting conditions. Overall, poly IC-exposed male offspring exhibited altered locomotor activity rhythms, reminiscent of individuals with neurodevelopmental disorders. In particular, we report increased (subjective) day activity across 3 different lighting conditions: 12 h of light, 12 h of dark (12:12LD), constant darkness (DD) and constant light. Further data analysis indicated that this was driven by increased activity in the beginning of the (subjective) day in 12:12LD and DD, and at the end of the day in 12:12LD. This effect was sex-dependent, as in utero poly IC exposure led overall to much milder alterations in locomotor activity rhythms in female offspring than in male offspring. We also confirmed that the observed behavioral impairments in adult poly IC-exposed offspring were not due to differences in maternal behavior. These data further our understanding of the link between circadian rhythm disruption and neurodevelopmental disorders and may have implications for mitigating risk to the disorders and/or informing the development of circadian-based therapies.

IMPACT OF INTRACORONARY ADENOSINE ADMINISTRATION DURING PRIMARY PERCUTANEOUS CORONARY INTERVENTION

Background: Currently myocardial reperfusion with p-PCI is the best treatment strategy for STEMI, However, myocardial perfusion at the cellular level remains impaired despite removal of coronary obstruction in up to 50% of STEMI patients Several methods have been evaluated to improve reperfusion, including heart rate reduction, aspiration thrombectomy and several pharmacological approaches, such as glycoprotein platelet inhibitors, adenosine and drugs able to dilate the microcirculation, seem to be most effective when locally delivered through IC injection, probably because this results in an increased drug bioavailability in the area at risk. Objective: the aim of our study was to clarify the efficacy of IC adenosine versus standard therapy only in STEMI patients undergoing p-PCI. Patients and methods: Selected 45 patients presented to emergency room by acute STEMI and were randomized into two groups: Control group (25 patients) and Patient group who received IC adenosine (20 patients). Both were evaluated during p-PCI by MBG and TIMI flow grade, after the procedure, both groups was evaluated by STR in ECG, cardiac enzymes at 0-6-12 hours, and echocardiography within 24 hours and after 40 days. Results: in patient group, TIMI flow was significantly better, incidence of ST resolution was significantly higher, and level of cardiac enzymes was significantly higher at 6 hours and significantly lower at 12 hours. Moreover, we found a larger increase in LVEF - and subsequently reduction in the incidence of heart failure, more improvement of MR, TR, PASP and TAPSE in patient group. Conclusion: This study clarified clinical benefits for IC adenosine in hard endpoints, such as TIMI flow, percentage of LVEF improvement, in patients undergoing p-PCI.

IMPACT OF INTRACORONARY ADENOSINE ADMINISTRATION DURING PRIMARY PERCUTANEOUS CORONARY INTERVENTION

Background: Currently myocardial reperfusion with p-PCI is the best treatment strategy for STEMI, However, myocardial perfusion at the cellular level remains impaired despite removal of coronary obstruction in up to 50% of STEMI patients Several methods have been evaluated to improve reperfusion, including heart rate reduction, aspiration thrombectomy and several pharmacological approaches, such as glycoprotein platelet inhibitors, adenosine and drugs able to dilate the microcirculation, seem to be most effective when locally delivered through IC injection, probably because this results in an increased drug bioavailability in the area at risk. Objective: the aim of our study was to clarify the efficacy of IC adenosine versus standard therapy only in STEMI patients undergoing p-PCI. Patients and methods: Selected 45 patients presented to emergency room by acute STEMI and were randomized into two groups: Control group (25 patients) and Patient group who received IC adenosine (20 patients). Both were evaluated during p-PCI by MBG and TIMI flow grade, after the procedure, both groups was evaluated by STR in ECG, cardiac enzymes at 0-6-12 hours, and echocardiography within 24 hours and after 40 days. Results: in patient group, TIMI flow was significantly better, incidence of ST resolution was significantly higher, and level of cardiac enzymes was significantly higher at 6 hours and significantly lower at 12 hours. Moreover, we found a larger increase in LVEF - and subsequently reduction in the incidence of heart failure, more improvement of MR, TR, PASP and TAPSE in patient group. Conclusion: This study clarified clinical benefits for IC adenosine in hard endpoints, such as TIMI flow, percentage of LVEF improvement, in patients undergoing p-PCI.

Abstract P2-20-03: Estrogen-depletion therapies prevent and delay progression of brain metastasis

Abstract Introduction: Triple negative breast cancers (TNBC, which lack estrogen receptor (ER), progesterone receptor and HER2), occur three times more frequently in premenopausal women and metastasize expeditiously to brain and lungs. Clinical and experimental evidence shows that estrogen, the main premenopausal ovarian hormone, promotes metastases of TNBC, through their action on the tumor microenvironment. We have shown that 17-β-Estradiol (E2) promotes experimental brain metastases (BM) of TNBC cells by inducing estrogen-receptor positive (ER+) astrocytes in the brain niche to secrete growth factors that promote cancer cell migration, invasion and growth. Given that 53% of pre-menopausal women with TNBC develops BM and 80% die within a year, it is now critical to define whether blocking estrogen signaling could have therapeutic value to prevent or delay progression of BM. Results: To assess whether E2-depletion could prevent brain metastatic colonization, brain trophic sublines of human TNBC (231BR), murine TNBC (4T1BR5), a non-selected TN cell line syngeneic to C57BL mice (E0771-GFP-luc) were intracardially (ic) injected two-days post-endocrine initiation, in ovariectomized (OVX) female mice treated with a) E2 (levels equivalent to those found in pre-menopausal women), b) placebo (OVX), or c) the aromatase-inhibitor (AI) letrozole (OVX + Letrozole) to block brain and peripheral E2-synthesis. BM were quantified histologically, via MRI or ex-vivo imaging of brains at euthanasia. E2-treated mice injected with E0771-GFP-luc cells showed an average of 6.6± 3.5 metastatic clusters per mice, compared to 3.6 ± 3.2 and 1.8 ± 1.8 in OVX mice alone or OVX + letrozole treated mice (a 3.6 fold increase, P=0.0013, E2 vs OVX + Let). Imaging of excised brains showed comparable results, with a mean total photon flux of 5.1 × 106 ± 5.3 × 106 in E2 vs 1.1 × 106 ± 1.5 × 106 in OVX + letrozole (P=0.0056). Similar preventive effects of E2-depletion were found in 4T1BR5 and 231BR-cell injected mice, using histological quantification and MRI as endpoints. To assess whether E2-depletion therapies could decrease brain metastatic progression in a therapeutic model mimicking the clinical setting, E0771-GFP-luc cells were injected in OVX-mice that had been supplemented with E2 and metastasis allowed to develop for 6 days. Mice were then randomized to a) E2, b) E2-withdrawal (E2WD) or c) E2WD+ letrozole, and then all mice were irradiated (whole brain irradiation, single-dose, 15 Gy) to mimic current standard of care for BMs. All mice were euthanized 10 days later, when E2-treated mice showed signs of CNS impairment. Mice that received E2WD+letrozole showed the lowest BM burden compared to mice that remained in E2-treatments, as assessed by histological quantification (0.7 ± 1.2 vs 6.5 ± 5.3 metastatic clusters, respectively, P=0.0132) and ex-vivo brain imaging (mean total-photon flux 3.4 × 105 ± 4.1 × 105 in E2WD + let vs 3.1 × 106 ± 1.4 × 106 in E2, P=0.037). Brain metastasis in TNBC often occur in women who also present with systemic metastatic disease, and E0771-GFP-luc cells can colonize multiple organs following ic injection. Thus, non-brain metastatic burden (systemic metastasis) was measured by in vivo imaging before euthanasia in this cohort. Unlike brain metastasis, E2WD or E2WD+letrozole showed no statistically significant effect on systemic metastatic burden (mean total flux 1.41 × 106 ± 0.9 × 106 in E2, vs 0.54 × 106 ± 2.6 × 106 in E2WD vs 0.79 × 106 ± 1.0 × 106 in E2WD+let, P=0.416), supporting the notion that brain-specific rather than systemic mechanisms drive progression of brain metastasis in response to E2. Conclusions: These studies have important clinical implications as they provide pre-clinical evidence that ovarian suppression in combination with AIs can prevent and delay progression of established brain metastasis in younger women with TNBC. Citation Format: Diana M. Cittelly, Maria J. Contreras-Zarate, Ricaurte A. Marquez-Ortiz, Nicole Day, Bebhinn Nagle, Ryan Ormond, Virginia F. Borges, Peter Kabos. Estrogen-depletion therapies prevent and delay progression of brain metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-20-03.

Ocular Intracameral Pharmacokinetics for a Cocktail of Timolol, Betaxolol, and Atenolol in Rabbits.

The mechanisms of drug clearance from the aqueous humor are poorly defined. In this study, a cocktail approach was used to simultaneously determine the pharmacokinetics of three β-blocker agents after intracameral (ic) injection into the rabbit eyes. Aqueous humor samples were collected and analyzed using LC-MS/MS to determine drug concentrations. Pharmacokinetic parameters were obtained using a compartmental fitting approach, and the estimated clearance, volume of distribution, and half-life values were the following: atenolol (6.44 μL/min, 687 μL, and 73.87 min), timolol (19.30 μL/min, 937 μL, and 33.64 min), and betaxolol (32.20 μL/min, 1421 μL, and 30.58 min). Increased compound lipophilicity (atenolol < timolol < betaxolol) resulted in higher clearance and volume of distributions in the aqueous humor. Clearance of timolol and betaxolol is about 10 times higher than the aqueous humor outflow, demonstrating the importance of other elimination routes (e.g., uptake to iris and ciliary body and subsequent elimination via blood flow).

Rapid activation of brain resident memory T cells following neurological insults

Abstract Brain tissue resident memory T cells (TRM) are an emerging lymphocyte of interest. While the phenotype and gene expression pattern of brain TRMs have recently been discussed, their function and response to neurologic insults is not yet understood. Using the described phenotype of TRMs (TCRB+, CD69+, CD4 or CD8+, CD103−, CD44+), we analyzed the reaction of this lymphocyte in the brain to various neurological insults. We found that physical insult induced by an intracranial(ic) injection of PBS resulted in increased TRM numbers within 24 hours post insult. This TRM population later decreased yet remained above baseline by 50 days post insult. Interestingly, TRM populations also increased in the brains of animals as a function of age, suggesting natural insults experienced by mice leads to an ever increasing TRM population in the brain. We then assessed the reaction of TRMs to virus in the brain. Mice infected ic with Theiler’s Murine Encephalomyelitis virus (TMEV) had a marked increase in brain TRMs one day later, which is 4 days prior to detection of traditional TMEV antigenspecific CD8 T cells. This implies TRMs respond to CNS viral infections prior to generation of virus antigen specific responses. Finally, we investigated the role of dendritic cell (DC) antigen presentation in generation of TRMs using our novel MHC class I conditional knockout mice. Conditional ablation of H-2Kb and H-2Db did not disrupt TRM populations in the brain. Our data is the first study to show: 1. Activation of TRMs in the brain preceded antigen specific cell infiltration and 2. MHC class I molecules on DCs are not required to generate brain resident TRMS. Understanding brain TRMs is crucial in elucidating their role in neurodegeneration and/or targeting them in CNS cancers.

447 Psychological distress in a sample of men with Peyronie's Disease attending a private urology clinic in Germany

Peyronie’s disease (PD) is a fibromatous disorder of the tunica albuginea of the penile corpus cavernosum with a genetic background and characterized by pain, plaque formation/calcification , penile curvature, and penile shortening. Although the epidemiological data on PD is inconsistent, most epidemiological studies report incidence rates of between 3 and 7 % cumulating in ages of 40-65 years. The objective of this study was to investigate psychological distress PD causes and which aspects were considered most distressing in a sample of men with PD presenting at a private Uro-Andrology clinic. The sample consisted of 119 men with PD (M age 51.6y) and 61 age-matched controls without PD. PD was assessed via clinical examination including color duplex ultrasonography and ic PGE1 injection test to measure the size, site(s) and number of plaques, as well as the degree of the penile curvature. In addition, comprehensive lab analyses were conducted and the degree of impairment by the condition, sexual functioning and psychological well-being were assessed using the Sexual Complaint Screener, the IIEF-EF, the AMS Scale, and the Peyronie’s Disease Questionnaire.

Development of Novel Patient-Derived Xenografts from Breast Cancer Brain Metastases.

Brain metastases are an increasing burden among breast cancer patients, particularly for those with HER2+ and triple negative (TN) subtypes. Mechanistic insight into the pathophysiology of brain metastases and preclinical validation of therapies has relied almost exclusively on intracardiac injection of brain-homing cells derived from highly aggressive TN MDA-MB-231 and HER2+ BT474 breast cancer cell lines. Yet, these well characterized models are far from representing the tumor heterogeneity observed clinically and, due to their fast progression in vivo, their suitability to validate therapies for established brain metastasis remains limited. The goal of this study was to develop and characterize novel human brain metastasis breast cancer patient-derived xenografts (BM-PDXs) to study the biology of brain metastasis and to serve as tools for testing novel therapeutic approaches. We obtained freshly resected brain metastases from consenting donors with breast cancer. Tissue was immediately implanted in the mammary fat pad of female immunocompromised mice and expanded as BM-PDXs. Brain metastases from 3/4 (75%) TN, 1/1 (100%) estrogen receptor positive (ER+), and 5/9 (55.5%) HER2+ clinical subtypes were established as transplantable BM-PDXs. To facilitate tracking of metastatic dissemination using BM-PDXs, we labeled PDX-dissociated cells with EGFP-luciferase followed by reimplantation in mice, and generated a BM-derived cell line (F2-7). Immunohistologic analyses demonstrated that parental and labeled BM-PDXs retained expression of critical clinical markers such as ER, progesterone receptor, epidermal growth factor receptor, HER2, and the basal cell marker cytokeratin 5. Similarly, RNA sequencing analysis showed clustering of parental, labeled BM-PDXs and their corresponding cell line derivative. Intracardiac injection of dissociated cells from BM-E22-1, resulted in magnetic resonance imaging-detectable macrometastases in 4/8 (50%) and micrometastases (8/8) (100%) mice, suggesting that BM-PDXs remain capable of colonizing the brain at high frequencies. Brain metastases developed 8–12 weeks after ic injection, located to the brain parenchyma, grew around blood vessels, and elicited astroglia activation characteristic of breast cancer brain metastasis. These novel BM-PDXs represent heterogeneous and clinically relevant models to study mechanisms of brain metastatic colonization, with the added benefit of a slower progression rate that makes them suitable for preclinical testing of drugs in therapeutic settings.

Oct1 and Oca-B Regulate the Myeloid Leukemic Phenotype

Oct1, encoded by the Pou2f1 gene, is a widely expressed transcription factor that is associated with metabolic control, stress response, stem cell phenotypes and malignant transformation. Oct1 associates with a number of cofactors including the activating cofactor OCA-B encoded by Pou2af1. OCA-B is normally expressed specifically in B cells, but is misexpressed in about 30% of AML cases in myeloid cells and is a poor prognostic indicator. We observed a conserved Oct1 binding site in the promoter of Cdx2, a homeodomain transcription factor that is normally expressed solely in the gastrointestinal tract of adult humans, but has been shown to misexpressed in blood cells in almost all cases of AML. Our study looks at the interaction of Oct1 with Cdx2, and the effects of Oct1 and OCAB-B knockout in a mouse model of AML.Using ChIP we determined that Oct1 is bound to the Cdx2 promoter in normal patient samples and AML patient samples. However, in normal patient samples a repressive cofactor (the NuRD complex) is also bound. In AML patient samples an activating cofactor (Jmjd1a) is bound. This suggests that in AML Oct1 is bound to Cdx2 and is associating with Jmjd1a, which can occur through ERK signaling or OCA-B expression, and activating expression of Cdx2.We next wanted to determine the effect of Oct1 knockout in AML using a MLL-AF9 mouse model. Our lab has developed a conditional Oct1 allele, as Oct1 germline knockout is embryonic lethal. We harvested bone marrow from Oct1+/fl;MX1-Cre, Oct1fl/D;MX1-Cre, and Oct1+/+;MX1-Cre mice and transduced the bone marrow with virus expressing MLL-AF9. Recipient mice were lethally irradiated and transplanted with the transduced bone marrow. Mice were injected with Poly IC at 5 weeks post-transplant to delete Oct1 foxed alleles. Mice that were wildtype for Oct1 developed leukemia around 7 weeks. Mice with one allele of Oct1 survived longer but still developed leukemia. Mice with no Oct1 survived longer still, but eventually suffered from bone marrow failure.In order to assess whether the lethality in Oct1 deficient mice with MLL-AF9-induced leukemia was due to the presence of the MLL-AF9 oncoprotein or if Oct1 deletion is lethal in adult mice under normal conditions, we treated non-leukemic mice that were Oct1+/fl;MX1-Cre or Oct1fl/D;MX1-Cre with Poly IC to delete Oct1 at 5 weeks. Following Poly IC injection, mice with and without Oct1 appeared normal and healthy by examination and by blood counts. At 20 weeks, all mice were injected with 5-flurouracil (5-FU). We observed that mice that were deficient in Oct1, namely Oct1fl/D;MX1-Cre mice injected with Poly IC, died within 2 weeks of 5-FU treatment. Put together, our data suggests that Oct1 is not necessary for normal hematopoiesis, as adult mice with no Oct1 appear normal under homeostatic conditions, but Oct1 is necessary for the hematopoietic proliferative stress response.The protective effect of Oct1 deletion against leukemia in mice suggested an important role for Oct1 in regulating the leukemic phenotype. However, since Oct1 deletion is also lethal in the leukemic context, we looked at the effect of deletion of OCA-B in leukemia. OCA-B germline deficient mice are viable and the only detectable phenotype is the lack of germinal center formation. We harvested OCA-B germline deficient and wildtype control bone marrow and followed the same protocol as outlined above with the transduction of the bone marrow with MLL-AF9. Mice with no OCA-B did not develop leukemia and had 100% survival. This striking result suggests that OCA-B could be a therapeutic target in MLL-AF9 AML. DisclosuresNo relevant conflicts of interest to declare.

Abstract A09: Development of novel breast cancer brain-metastases-derived xenografts and cell lines

Abstract Symptomatic brain metastases develop in 10 to 16% of patients with metastatic breast cancer and the prognosis of these patients is dismal with a mortality rate of near 80% within 1 year of diagnosis. Understanding the mechanisms by which breast cancer cells adapt to the brain microenvironment is key to design therapies that could be used to prevent or treat brain metastases. To date, the field has relied almost exclusively on intracardiac (ic) injection of brain-homing cells derived from highly aggressive triple negative (TN) MDA-MB-231 and HER2+ BT474 (BT474BR) cell lines to study these interactions. Although these are well characterized models, these are far from representing the heterogeneity observed in breast tumors and their metastases. Purpose: The goal of this study was to develop and characterize novel human brain metastases patient-derived xenografts (BM-PDX) to study the biology of brain metastases and to serve as tools for testing novel therapeutic approaches. We have also developed a labeling method to tag these PDXs with traceable markers for in vivo studies. Experimental procedures: Metastatic tissue was removed from brain parenchyma during surgery (n=5) or from cerebral spinal fluid (CSF) via catheter (n=1) from consenting donors harboring TN (n=3) or HER2+ (n=3) metastatic breast cancer. Tissue was immediately implanted in the mammary fat pad of NOD/SCID/GAMMA female mice and tumor expanded as solid tumors (BM-PDX). Upon reaching a volume of &amp;gt;200 mm3, partitioned pieces were transplanted into new recipient mice and growth kinetics measured. In order to track metastatic spread using PDXs, we developed a protocol to label PDX-dissociated cells with GFP-luciferin followed by re-implantation in mice. Tumor sections before and after labeling were immunostained for hormone receptors (estrogen, progesterone and androgen receptors –ER, PR, AR), HER2, and the basal cell marker cytokeratin 5 (CK5). These were scored by a pathologist. Dissociated cells from an established BM-PDX were cultured under different conditions in vitro, to generate a BM-derived cell line. Gene expression profiling was performed in some PDX before and after GFP-luciferase labeling, and in derived cell lines. Dissociated cells from BM-PDXs (E22-1) were injected intracardially (n=5) to measure their ability to colonize the brain in an experimental model of brain metastasis. Summary: Three of 3 TN brain metastases were successfully established as transplantable PDXs, while 0 of 3 HER2+ tumors grew in mice. Immunohistochemical analyses demonstrated that BM-PDXs retain morphology and markers comparable to those of donor samples. Labeled tumors grew in the mammary fat pad without spontaneous metastatic spread as measured via in vivo imaging (IVIS). Intracardiac injection of dissociated cells from BM-PDX E22-1 resulted in micro or macrometastases in 5/5 (100%) injected mice, indicating that BM-PDXs remain capable of colonizing the organ of origin. Metastases developed in brain parenchyma, surrounding vessels, with associated microglia and astroglia activation, similar to what is observed in human brain metastases. BM-PDX dissociated cells were cultured under different conditions to derive BM-cell lines. Two cell lines have been derived from a TN BM-PDX (F2-6) after in vitro culture using brain-like media or primary cell media. RNAseq analyses to fully characterize the genetic makeup of BM-PDX and cell line derivatives are currently in progress. Conclusions: We have developed novel PDXs derived from breast cancer brain metastases, which retain their ability to colonize the brain when disseminated in circulation. These novel GFP-luciferase labeled xenografts and cell lines, represent unique tools to study brain metastasis biology and to test novel therapeutic treatments for brain metastasis in clinically relevant models. Citation Format: Colton Hanna, Britta M. Jacobsen, Carol A. Sartorius, Peter Kabos, Kevin Lillehei, David R. Ormond, Michael Graner, Virginia F. Borges, Diana M. Cittelly. Development of novel breast cancer brain-metastases-derived xenografts and cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A09.

Abstract P4-04-04: Estrogen signaling through astrocytes promotes migration and invasion of ER-negative brain metastatic breast cancer cells

Abstract Approximately 16% of patients with breast cancer develop symptomatic brain metastases (BMs) and the majority [∼80%] will die subsequently. Symptomatic BMs are more prevalent in breast tumors overexpressing Human Epidermal Growth Factor Receptors (HER1, EGFR) or 2 (HER2) and triple-negative (TN) tumors (lacking estrogen receptor (ER), progesterone receptor (PR) and HER2). Although the majority of breast cancer cells colonizing the brain lack ER, central nervous system (CNS) metastases are more frequent in pre-menopausal women with high levels of circulating estrogens. Since astrocytes are ER+ cells that surround and infiltrate brain metastases, we hypothesize that estrogens act in a paracrine manner on reactive astrocytes to promote BMs. Results: To determine the effect of estrogen in brain colonization, a brain-seeking sub-line of human MDA-MB-231 TN breast carcinoma cells (231Br) cells were injected intracardially in ovariectomized female nude mice supplemented with placebo (n=5) or 1mg 17-βestradiol (E2) pellets (n=5), and metastases were detected by gadolinium-enhanced magnetic resonance imaging (MRI) 5 weeks later. 100% (5/5) of mice from E2 group showed large and multiple BM when compared to 3/5 (60%) of placebo mice. Further, E2-treated mice showed decreased survival (41.6%, n=12) by 3 weeks following IC injection of 231Br cells compared to E2-depleted mice (84%, n=12) (P=0.03). Since astrocytes are the most abundant brain cell type expressing ERs, we focused on their interaction with 231BR cells. Using global gene expression arrays and RT-PCR we found that E2 upregulated Egf and Tgf-a in primary mouse astrocytes and human astrocytes (derived from neural stem cells), and that anti-estrogens blocked this effect. Concentrated conditioned media (CM) from E2-treated astrocytes increased P-EGFR levels in 231Br cells compared to vehicle (EtOH), or E2+ICI astrocytic CM. Moreover, CM from E2-treated astrocytes significantly increased migration and invasion of 231Br cells as compared to EtOH-treated CM, and ERs inhibitors abolish this effect. Treatment of 231Br cells with lapatinib abolished EGFR activation in response to E2-astrocytic CM, and decreased migration and invasion of 231BR cells. These data suggest that EGFR-ligands upregulated by E2 in astrocytes activated brain metastatic cells-EGFR resulting in increased migration and invasion. To identify the mechanisms of increased migration and invasion we performed global gene expression profiling of 231Br cells co-cultured with E2- or EtOH-stimulated-astrocytes. Co-culture with E2-treated astrocytes significantly increased expression of metastatic mediators Matrix-metalloproteinase-9 (MMP9) and S100 Calcium-binding protein A4 (S100A4) in 231Br cells, and treatment of astrocytes with ER inhibitors abolished this effect. Furthermore, MMP9 or S100A4 knockdown in 231Br cells significantly decreased migration and/or invasion in response to E2-astrocytic CM, suggesting that MMP9 and S100A4 are functional mediators of the paracrine effect of E2. Conclusion: These studies suggest that EGFR activation by astrocytic ligands is at least one mechanism by which E2 contributes to the promotion of BM of ER- breast cancer cells and suggests a novel role for ER in breast cancer BM. Citation Format: Diana M Cittelly, Hazel Cruz, Natalie J Serkova, Borges F Virginia, Kabos Peter, Steeg S Patricia, Sartorius A Carol. Estrogen signaling through astrocytes promotes migration and invasion of ER-negative brain metastatic breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-04.

WE‐E‐108‐04: X‐Ray Fluorescence Imaging Guided Microbeam Radiation Therapy

Purpose: We demonstrate the feasibility of x‐ray fluorescence for quick and accurate image guidance for targeted irradiation of small animal tumor models in Microbeam Radiation Therapy (MRT). Methods: A reduced‐dose treatment beam excites x‐ray fluorescence from contrast agents preferentially‐accumulating at tumor sites. Intensity profiles of the fluorescence signal are mapped as the mouse injected with the contrast agent is moved through the field of view. The tumor location is identified as the regions with high fluorescence intensities. For this feasibility study, our compact small‐animal MRT system and a mouse head phantom were used. The MRT device uses a carbon nanotube (CNT) field emission x‐ray source array operating at 160kVp and adjustable‐width external collimator to generate microbeam radiation at a microbeam dose rate of ∼1Gy/min. A phantom consisting of an acrylic rod inserted into a mouse skull accurately represents the attenuation of the head. The rod contains ∼1.5mm diameter holes separated by 4mm, filled with 12mg/mL iodine solution, similar to the reported tumor concentration from IC injection. The phantom was translated through the treatment beam in 600um steps, 20 seconds per step. The fluorescence signal was recorded using a slit‐collimated spectrum analyzer. Results: Processed spectra displayed fluorescent intensity peaks corresponding with locations of iodine solution in the phantom. The FWHMs of these peaks are 1.78mm and 1.72mm. The actual diameters of corresponding regions are 1.56mm and 1.47mm, indicating ∼250um uncertainty. Conclusion: Preliminary results demonstrated feasibility of x‐ray fluorescence for guidance in MRT. The acquisition time for a 15mm head phantom is ∼8 minutes, within the reported retention time of the contrast agent. ∼250um uncertainty is comparable to results from our in vivo brain tumor‐bearing mouse experiment using MRI and x‐ray image guidance. The x‐ray fluorescent method is substantially faster. The process will be further optimized and validated with in‐vivo studies.

SAT0061 No migration of injected mesenchymal stem cells to arthritic joints

BackgroundMSC’s are adult stem cells present in many tissues, potently immunosuppressive, shown to specifically migrate to damaged areas. MSC’s have been shown to diminish arthritis in animal models although the...

Analysis of Erectile Responses to BAY 41-8543 and Muscarinic Receptor Stimulation in the Rat

Soluble guanylate cyclase (sGC) is the receptor for nitric oxide (NO) and in pathophysiologic conditions where NO formation or bioavailability is impaired, erectile dysfunction (ED) occurs. The aim of this study was to investigate erectile responses to the sGC stimulator BAY 41-8543 in physiologic and pathophysiologic conditions. Increases in intracavernosal pressure (ICP) in response to intracavernosal (ic) injections of BAY 41-8543 were investigated in the anesthetized rat. Increases in ICP/MAP in response to ic injections of BAY 41-8543 and the interaction of BAY 41-8543 with exogenous and endogenously released NO were investigated and the effect of the sGC stimulator on cavernosal nerve injury was assessed. The mechanism of the increase in ICP/MAP in response to ic injection of acetylcholine was investigated. The ic injections of BAY 41-8543 increased ICP/MAP and the duration of the response. BAY 41-8543 was less potent than sodium nitroprusside (SNP) and ic injections of BAY 41-8543 and SNP produced a larger response than the algebraic sum of responses to either agent alone. Simultaneous ic injection of BAY 41-8543 and cavernosal nerve stimulation produced a greater response than either intervention alone. Atropine and cavernosal nerve crush injury decreased the response to nerve stimulation and ic injection of BAY 41-8543 restored the response. These data show that BAY 41-8543 has significant erectile activity and can synergize with exogenous and endogenously released NO. This study shows that atropine and nerve crush attenuate the response to cavernosal nerve stimulation and that BAY 41-8543 can restore the response. The results with atropine, L-NAME and hexamethonium indicate that the response to ic injection of acetylcholine is mediated by muscarinic receptors and the release of NO with no significant role for nicotinic receptors. These results suggest that BAY 41-8543 would be useful in the treatment of ED.

119 Size-dependent retention of stem cells following intracoronary injection

BackgroundIntracoronary (IC) injection of bone marrow mononuclear cells (BMMNCs) is a promising treatment for heart failure. However, therapeutic effects in clinical studies have been small and a principle reason may...

9‐Hydroxycanthin‐6‐One Induces Penile Erection and Delays Ejaculation

Eurycoma longifolia Jack (Simaroubaceae) has the reputation as a male aphrodisiac because it is claimed to increase virility and sexual prowess. Nevertheless, whether or not E. longifolia regulates directly the muscle tone of corpus cavernosa and/or seminal vesicle (SV) remains unclear. Even until now, the compositions that could account for its aphrodisiac property are still unknown We examined the effect of 9-hydroxycanthin-6-one (9-HC-6-one), a β-carboline alkaloid isolated from E. longifolia, on penile erection and ejaculation, and further elucidated the mechanism of action. 9-HC-6-one induces penile erection and delays ejaculation. Drug's effect was studied on rat corpus cavernosum (CC) and SV in vitro, and on the changes in intracavernosal pressure (ICP) after IC injection and intraluminal pressure (ILP) of the SV after hypogastric nerve stimulation (HNS), respectively. 9-HC-6-one relaxed significantly phenylephrine (PE)-precontracted CC. Such response was not attenuated by endothelium disruption, N(G) -nitro-L-arginine methyl ester, or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one treatment, suggesting that a nitric oxide/cyclic guanosine monophosphate-dependent pathway was precluded. 9-HC-6-one attenuated PE-induced contraction by blocking cell surface and internal calcium channels with a higher potency for internal calcium release. This compound also antagonized calcium-evoked contraction in Ca2+ -free, high K+ -depolarizing condition, suggesting that interfering with the entry of calcium through voltage-dependent channels also contributed to 9-HC-6-one-induced corporal relaxation. After IC application of 9-HC-6-one, a significant rise in ICP was observed as compared with the application of normal saline. 9-HC-6-one relaxed significantly norepinephrine (NE)- and KCl-precontracted SV, and antagonized NE-induced oscillatory contraction as potent as clomipramine. Finally, the HNS-evoked increase in ILP was dose-dependently repressed after challenge by 9-HC-6-one. 9-HC-6-one might be the active component that contributed to the aphrodisiac effect of E. longifolia by antagonizing the smooth muscle tone of CC as well as SV probably through interfering with Ca2+ mobilization.

Endogenous orexin-A in the brain mediates 2-deoxy-d-glucose-induced stimulation of gastric motility in freely moving conscious rats

Increasing evidence has indicated that brain orexin plays a vital role in the regulation of gastrointestinal (GI) physiology such as gastric acid secretion and GI motility. The aim of this study was to elucidate the effects and mechanisms of orexin on gastric motility in non-fasted rats. In this study, we recorded intraluminal gastric pressure waves in freely moving conscious rats with a manometric catheter located in the antrum. We assessed the area under the manometric trace as the motor index (MI), and compared its values for 1 h before and after drug administration. Intracisternal (ic) injection of orexin-A (10 μg) significantly increased the MI, but intraperitoneal (ip) injection did not have any effect. Pretreatment of ip injection of atropine significantly blocked the orexin-A-induced stimulation of gastric motility. Intravenous injection of 2-deoxy-D-glucose (2-DG, 200 mg/kg), a central vagal stimulant, significantly increased the MI. The ic injection of SB-334687 (40 μg), a selective orexin-A antagonist, did not modify the basal MI, but this antagonist significantly suppressed the stimulant action of 2-DG. These results suggest that endogenous orexin-A in the brain is involved in the vagal-dependent stimulation of gastric contractions.

Recruitment of Intracavernously Injected Adipose-Derived Stem Cells to the Major Pelvic Ganglion Improves Erectile Function in a Rat Model of Cavernous Nerve Injury

BackgroundIntracavernous (IC) injection of stem cells has been shown to ameliorate cavernous-nerve (CN) injury-induced erectile dysfunction (ED). However, the mechanisms of action of adipose-derived stem cells (ADSC) remain unclear. ObjectivesTo investigate the mechanism of action and fate of IC injected ADSC in a rat model of CN crush injury. Design, setting, and participantsSprague-Dawley rats (n=110) were randomly divided into five groups. Thirty-five rats underwent sham surgery and IC injection of ADSC (n=25) or vehicle (n=10). Another 75 rats underwent bilateral CN crush injury and were treated with vehicle or ADSC injected either IC or in the dorsal penile perineural space. At 1, 3, 7 (n=5), and 28 d (n=10) postsurgery, penile tissues and major pelvic ganglia (MPG) were harvested for histology. ADSC were labeled with 5-ethynyl-2-deoxyuridine (EdU) before treatment. Rats in the 28-d groups were examined for erectile function prior to tissue harvest. MeasurementsIC pressure recording on CN electrostimulation, immunohistochemistry of the penis and the MPG, and number of EdU-positive (EdU+) cells in the injection site and the MPG. Results and limitationsIC, but not perineural, injection of ADSC resulted in significantly improved erectile function. Significantly more EdU+ ADSC appeared in the MPG of animals with CN injury and IC injection of ADSC compared with those injected perineurally and those in the sham group. One day after crush injury, stromal cell-derived factor-1 (SDF-1) was upregulated in the MPG, providing an incentive for ADSC recruitment toward the MPG. Neuroregeneration was observed in the group that underwent IC injection of ADSC, and IC ADSC treatment had beneficial effects on the smooth muscle/collagen ratio in the corpus cavernosum. ConclusionsCN injury upregulates SDF-1 expression in the MPG and thereby attracts intracavernously injected ADSC. At the MPG, ADSC exert neuroregenerative effects on the cell bodies of injured nerves, resulting in enhanced erectile response.