Spontaneous Mouse Model Research Articles

STING expression and response to treatment with STING ligands in premalignant and malignant disease.

Human papilloma virus positive (HPV+) tumors represent a large proportion of anal, vulvar, vaginal, cervical and head and neck squamous carcinomas (HNSCC) and late stage invasive disease is thought to originate from a premalignant state. Cyclic dinucleotides that activate STimulator of INterferon Genes (STING) have been shown to cause rapid regression of a range of advanced tumors. We aimed to investigate STING ligands as a novel treatment for papilloma. We tested therapies in a spontaneous mouse model of papilloma of the face and anogenital region that histologically resembles human HPV-associated papilloma. We demonstrate that STING ligands cause rapid regression of papilloma, associated with T cell infiltration, and are significantly more effective than Imiquimod, a current immunotherapy for papilloma. In humans, we show that STING is expressed in the basal layer of normal skin and lost during keratinocyte differentiation. We found STING was expressed in all HPV-associated cervical and anal dysplasia and was strongly expressed in the cancer cells of HPV+ HNSCC but not in HPV-unrelated HNSCC. We found no strong association between STING expression and progressive disease in non-HPV oral dysplasia and oral pre-malignancies that are not HPV-related. These data demonstrate that STING is expressed in basal cells of the skin and is retained in HPV+ pre-malignancies and advanced cancers, but not in HPV-unrelated HNSCC. However, using a murine HNSCC model that does not express STING, we demonstrate that STING ligands are an effective therapy regardless of expression of STING by the cancer cells.

Nicotinic Acetylcholine Receptor Subtype Alpha-9 Mediates Triple-Negative Breast Cancers Based on a Spontaneous Pulmonary Metastasis Mouse Model.

Triple-negative breast cancer (TNBC) subtype is associated with poor prognosis and a high risk of recurrence-related death in women. Despite the aggressiveness of TNBCs, targeted TNBC therapy is not yet available in the clinic. To overcome this challenge, we generated highly metastatic TNBC cells (LM) derived from metastasized lung cells via a serial spontaneous pulmonary metastasis animal model to identify targetable molecules for attenuating the progression of TNBC metastasis. Gene analysis of primary tumor (P), first-round (1LM) and second-round (2LM) metastasized lung cells revealed that mesenchymal-related genes were significantly expressed in LM cells, especially in 2LM cells. Interestingly, α9-nAChR gene expression was also dramatically induced in LM cells, confirming our previous finding that α9-nAChR plays important roles in receptor-mediated carcinogenic signals in human breast cancer development. Using α9-nAChR as a biomarker, we transfected 2LM cells with CRISPR/Cas9 lentivirus targeting the α9-nAChR genomic region (2LM-α9-nAChR-null), showing that mesenchymal markers and the migration and invasion abilities of 2LM cells were significantly attenuated in 2LM-α9-nAChR-null cells both in vitro and in vivo. In addition, the high efficiency of editing the α9-nAChR gene using a CRISPR/Cas9 lentivirus was demonstrated by gene sequencing, genomic indel frequency and protein expression analyses. Collectively, these results confirmed those of our previous study that advanced-stage breast tumors are associated with substantially higher levels of α9-nAChR gene expression, indicating that α9-nAChR expression is essential for mediating TNBC metastasis during cancer development and may potentially act as a biomarker for targeted therapy in clinical investigations.

Developing chemotherapy for diffuse pontine intrinsic gliomas (DIPG).

Prognosis of diffuse intrinsic pontine glioma (DIPG) is poor, with a median survival of 10 months after radiation. At present, chemotherapy has failed to show benefits over radiation. Advances in biotechnology have enabled the use of autopsy specimens for genomic analyses and molecular profiling of DIPG, which are quite different from those of supratentorial high grade glioma. Recently, combined treatments of cytotoxic agents with target inhibitors, based on biopsied tissue, are being examined in on-going trials. Spontaneous DIPG mice models have been recently developed that is useful for preclinical studies. Finally, the convection-enhanced delivery could be used to infuse drugs directly into the brainstem parenchyma, to which conventional systemic administration fails to achieve effective concentration. The WHO glioma classification defines a diffuse midline glioma with a H3-K27M-mutation, and we expect increase of tissue confirmation of DIPG, which will give us the biological information helping the development of a targeted therapy.

De Novo Lipid Synthesis Facilitates Gemcitabine Resistance through Endoplasmic Reticulum Stress in Pancreatic Cancer.

Pancreatic adenocarcinoma is moderately responsive to gemcitabine-based chemotherapy, the most widely used single-agent therapy for pancreatic cancer. Although the prognosis in pancreatic cancer remains grim in part due to poor response to therapy, previous attempts at identifying and targeting the resistance mechanisms have not been very successful. By leveraging The Cancer Genome Atlas dataset, we identified lipid metabolism as the metabolic pathway that most significantly correlated with poor gemcitabine response in pancreatic cancer patients. Furthermore, we investigated the relationship between alterations in lipogenesis pathway and gemcitabine resistance by utilizing tissues from the genetically engineered mouse model and human pancreatic cancer patients. We observed a significant increase in fatty acid synthase (FASN) expression with increasing disease progression in spontaneous pancreatic cancer mouse model, and a correlation of high FASN expression with poor survival in patients and poor gemcitabine responsiveness in cell lines. We observed a synergistic effect of FASN inhibitors with gemcitabine in pancreatic cancer cells in culture and orthotopic implantation models. Combination of gemcitabine and the FASN inhibitor orlistat significantly diminished stemness, in part due to induction of endoplasmic reticulum (ER) stress that resulted in apoptosis. Moreover, direct induction of ER stress with thapsigargin caused a similar decrease in stemness and showed synergistic activity with gemcitabine. Our in vivo studies with orthotopic implantation models demonstrated a robust increase in gemcitabine responsiveness upon inhibition of fatty acid biosynthesis with orlistat. Altogether, we demonstrate that fatty acid biosynthesis pathway manipulation can help overcome the gemcitabine resistance in pancreatic cancer by regulating ER stress and stemness. Cancer Res; 77(20); 5503-17. ©2017 AACR.

Postpartum Lactation-Mediated Behavioral Outcomes and Drug Responses in a Spontaneous Mouse Model of Obsessive-Compulsive Disorder.

Using a spontaneous mouse model of obsessive-compulsive disorder (OCD), the current study evaluated the influence of postpartum lactation on the expression of compulsive-like behaviors, SSRI effectiveness, and the putative role of oxytocin and dopamine in mediating these lactation specific behavioral outcomes. Compulsive-like lactating mice were less compulsive-like in nest building and marble burying and showed enhanced responsiveness to fluoxetine (50 mg/kg) in comparison to compulsive-like nonlactating and nulliparous females. Lactating mice exhibited more anxiety-like behavior in the open field test compared to the nulliparous females, while chronic fluoxetine reduced anxiety-like behaviors. Blocking the oxytocin receptor with L368-899 (5 mg/kg) in the lactating mice exacerbated the compulsive-like and depression-like behaviors. The dopamine D2 receptor (D2R) agonist bromocriptine (10 mg/kg) suppressed marble burying, nest building, and central entries in the open field, but because it also suppressed overall locomotion in the open field, activation of the D2R receptor may have inhibited overall activity nonspecifically. Lactation- and fluoxetine-mediated behavioral outcomes in compulsive-like mice, therefore, appear to be partly regulated by oxytocinergic mechanisms. Serotonin immunoreactivity and serum levels were higher in lactating compulsive-like mice compared to nonlactating and nulliparous compulsive-like females. Together, these results suggest behavioral modulation, serotonergic alterations, and changes in SSRI effectiveness during lactation in compulsive-like mice. This warrants further investigation of postpartum events in OCD patients.

Endogenous glutamine decrease is associated with pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDAC) is becoming the second leading cause of cancer-related death in the Western world. The mortality is very high, which emphasizes the need to identify biomarkers for early detection. As glutamine metabolism alteration is a feature of PDAC, its in vivo evaluation may provide a useful tool for biomarker identification. Our aim was to identify a handy method to evaluate blood glutamine consumption in mouse models of PDAC. We quantified the in vitro glutamine uptake by Mass Spectrometry (MS) in tumor cell supernatants and showed that it was higher in PDAC compared to non-PDAC tumor and pancreatic control human cells. The increased glutamine uptake was paralleled by higher activity of most glutamine pathway-related enzymes supporting nucleotide and ATP production. Free glutamine blood levels were evaluated in orthotopic and spontaneous mouse models of PDAC and other pancreatic-related disorders by High-Performance Liquid Chromatography (HPLC) and/or MS. Notably we observed a reduction of blood glutamine as much as the tumor progressed from pancreatic intraepithelial lesions to invasive PDAC, but was not related to chronic pancreatitis-associated inflammation or diabetes. In parallel the increased levels of branched-chain amino acids (BCAA) were observed. By contrast blood glutamine levels were stable in non-tumor bearing mice. These findings demonstrated that glutamine uptake is measurable both in vitro and in vivo. The higher in vitro avidity of PDAC cells corresponded to a lower blood glutamine level as soon as the tumor mass grew. The reduction in circulating glutamine represents a novel tool exploitable to implement other diagnostic or prognostic PDAC biomarkers.

Basophils contribute to pristane-induced Lupus-like nephritis model

Lupus nephritis (LN), one of the most severe outcomes of systemic lupus erythematosus (SLE), is initiated by glomerular deposition of immune-complexes leading to an inflammatory response and kidney failure. Autoantibodies to nuclear antigens and autoreactive B and T cells are central in SLE pathogenesis. Immune mechanisms amplifying this autoantibody production drive flares of the disease. We previously showed that basophils were contributing to LN development in a spontaneous lupus-like mouse model (constitutive Lyn−/− mice) and in SLE subjects through their activation and migration to secondary lymphoid organs (SLOs) where they amplify autoantibody production. In order to study the basophil-specific mechanisms by which these cells contribute to LN development, we needed to validate their involvement in a genetically independent SLE-like mouse model. Pristane, when injected to non-lupus-prone mouse strains, induces a LN-like disease. In this inducible model, basophils were activated and accumulated in SLOs to promote autoantibody production. Basophil depletion by two distinct approaches dampened LN-like disease, demonstrating their contribution to the pristane-induced LN model. These results enable further studies to decipher molecular mechanisms by which basophils contribute to lupus progression.

Abstract 3006: Oncogenic activity of H3.3 K27M in a spontaneous DIPG mouse model

Abstract Pediatric diffuse high-grade gliomas (HGG) cause a devastatingly poor outcome. Diffuse intrinsic pontine gliomas (DIPG) are brainstem HGGs found almost exclusively in children and represent approximately 50 percent of all pediatric HGGs. Recurrent, clonal somatic mutations in histone H3 are a molecular hallmark that distinguish the genetic mechanisms underlying pediatric and adult HGG, and indicate a strong link between epigenetic dysfunction and pediatric brain tumorigenesis. H3 K27M mutations found in nearly 80 percent of DIPGs and over half of non-brainstem HGGs occurring in thalamus or other midline structures, induce a dominant loss of genome-wide H3K27me3. In contrast, mutually exclusive H3.3 G34R/V mutations occur in approximately 14 percent of cortical HGG tumors in older children through young adulthood suggesting a distinct developmental origin compared to K27M mutant tumors. DNA methylation and gene expression patterns distinguish G34R/V and K27M tumors, but the oncogenic mechanisms and reasons for brain region selectivity of histone mutations remain uncertain. To study these mutations in the developing mammalian brain and investigate how they contribute to oncogenesis, we generated conditionally activated, epitope-tagged knock-in mice to express K27M, G34R or non-mutated H3.3 proteins from the endogenous H3f3a locus. Untransformed embryonic neural precursor or astrocyte cells expressing H3.3 K27M demonstrated H3K27me3 loss and a growth advantage in vitro with brain region-specific, stage-dependent expression and epigenetic signatures. DIPGs frequently harbor genetic alterations in TP53 and PDGFRα in addition to H3.3 K27M, and mice with induced brain-specific Trp53 deletion plus mutated PDGFRαV544ins expression developed spontaneous HGG in vivo. Induction of H3.3 K27M significantly accelerated PDGFRαV544ins;Trp53 knockout HGG formation involving the brainstem. Importantly, the non-mutated H3.3 epitope-tagged protein did not affect tumor burden or latency. The H3.3 K27M tumors recapitulate characteristics observed in human DIPG including histopathological features, strong nuclear K27M expression and robust loss of H3K27me3 throughout the tumors. We relate the genetic, epigenetic and transcriptional landscapes of these models to primary pediatric DIPG to help identify their developmental origins and oncogenic mechanisms downstream of histone H3.3 K27M. Citation Format: Jon D. Larson, Lawryn H. Kasper, Gang Wu, Yiping Fan, David Finkelstein, Hongjian Jin, Barbara S. Paugh, Andre B. Silveira, Beisi Xu, Xiaoyan Zhu, Junyuan Zhang, Helen R. Russell, Peter J. McKinnon, David W. Ellison, Jinghui Zhang, Suzanne J. Baker. Oncogenic activity of H3.3 K27M in a spontaneous DIPG mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3006. doi:10.1158/1538-7445.AM2017-3006

Abstract 1844: Investigating the role of amphiregulin in breast cancer

Abstract Breast cancer is the most prevalent cancer in women worldwide and afflicts more than 3.1 million women in the US alone. The epidermal growth factor receptor (EGFR) is frequently overexpressed in breast cancer and is associated with poor clinical outcome. Several therapies involving anti-EGFR agents have been developed to target breast cancer. However, clinical trials have shown mixed outcomes with these therapies. Amphiregulin (AREG) is a ligand for EGFR that mediates estrogen-induced mammary ductal morphogenesis during development. Expression of AREG is enriched in invasive breast carcinomas, particularly ERα-positive breast tumors. Studies have shown that AREG increases the in vitro invasiveness of breast cancer cells. Furthermore, suppression of AREG expression in transformed human breast epithelial cells reduces tumor formation when injected in nude mice. These observations suggest that AREG plays an important role in breast cancer development and progression. To investigate the role of AREG in breast cancer development and progression, we utilized a spontaneous mammary tumor mouse model where oncogenesis is driven by expression of the polyoma middle T oncoprotein (PyMT) through the mouse mammary tumor virus (MMTV) promoter. We have crossed AREG knockout mice with the MMTV-PyMT mice to generate AREG KO mice that spontaneously form mammary tumors. Tumors in AREG KO PyMT mice initially grow more quickly but eventually reach 2 cm in diameter at a relatively time as compared to tumors formed in AREG WT PyMT mice. Histologically, AREG KO tumors appear more differentiated and display a greater proportion of tumor stroma. This prompted us to further hypothesize that AREG may have diverse effects in the tumoral or stromal compartment of developing tumors. To individually assess the effects of AREG knockout in the tumor cells and the stroma, we utilized a transplantation model in which we prepared tumor chunks from AREG WT and KO PyMT tumors and transplanted them into AREG WT and KO mice. Through this model, we have found that AREG expression in the tumor has a greater impact on tumor growth and progression than the tumor stroma. Regardless of the AREG status of the recipient mice, AREG KO tumors display a significant growth impairment and reduced transplant efficiency. In summary, these results demonstrate that AREG is important for mammary tumor progression. Citation Format: Serena P. Chiang, George S. Karagiannis, John S. Condeelis, Jeffrey E. Segall. Investigating the role of amphiregulin in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1844. doi:10.1158/1538-7445.AM2017-1844

Abstract 4229: Metabolically active macrophages: A novel link between obesity and TNBC

Abstract Triple negative breast cancer (TNBC) patients have an extremely poor prognosis due to their high metastatic potential and lack of targeted drug therapies. Emerging epidemiological data suggest that obesity is strongly linked to the incidence and severity of TNBC. However, mechanisms by which obesity potentiates TNBC progression are unclear. Adipose tissue macrophages (ATMs) are an attractive mechanistic link between obesity and TNBC because they are the predominant type of macrophage in the breast during early tumorigenesis and obesity causes accumulation of ATMs. However, during obesity, the macrophage phenotype was reported to switch from an apparent pro-tumorigenic M2-like phenotype to an apparent anti-tumorigenic M1-like phenotype. This result raises a paradox: how does obesity promote tumor progression if it activates an anti-tumorigenic macrophage phenotype? Here we show that metabolic dysfunction promotes a mechanistically distinct pro-inflammatory phenotype (metabolic activation; MMe) in breast adipose tissue macrophages isolated from obese women. These ATMs express cell surface markers of MMe (CD36, ABCA1) but not M1 (CD38, CD319, CD274) macrophages. We further demonstrate that pretreating TNBC cells with conditioned media derived from MMe but not M1 macrophages promotes mammosphere formation as well as invasion in vitro, and intravasation of cancer cells into the blood in vivo. Moreover, pre-treated cancer cells showed a two-fold increase in the expression of stem-like cell markers (OCT4, SOX2 and NANOG). Remarkably, we saw the same effect on ‘stemness’ of tumor cells in a spontaneous TNBC mouse model when fed a 60% high fat diet and a significant increase in tumor incidence in obese mice compared to lean mice in a syngeneic mouse model. We further show that blocking inflammatory cytokines expression in MMe(s) using genetic knockouts inhibits the ability of MMe(s) to promote both mammosphere formation and invasion. These findings suggest that obesity-induced changes to mammary adipose tissue reprogram macrophages to an MMe phenotype that potentiates TNBC initiation and metastasis via inflammatory cytokines. A comprehensive understanding of the signaling mechanism(s) involved in metabolic activation of mammary ATMs would enable development of directed therapies towards this specific pro-tumorigenic macrophage phenotype, thereby leaving the immune system of cancer patients intact. Citation Format: Payal Tiwari, Kelly Schoenfelt, Ariane Blank, Chang Cui, Marsha Rich Rosner, Lev Becker. Metabolically active macrophages: A novel link between obesity and TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4229. doi:10.1158/1538-7445.AM2017-4229

Abstract 942: CXCR2 acts as a checkpoint regulator of the pancreatic stellate cells activity within pancreatic cancer tumor microenvironment

Abstract Pancreatic cancer (PC) is a grotesque disease featured by an inflamed complex tumor microenvironment (TME) that contribute to advancing tumor progressing. Oncogenic K-ras, the most common mutation in PC, amplifies inflammation within TME through overexpression of multiple immune factors such as CXCR2 and its ligands. K-ras induced overexpression of CXCR2 axis, made CXCR2 seem intuitively a good target for PC treatment. In KC, a spontaneous PC mouse model with oncogenic K-ras, genetic ablation of CXCR2 caused anti-tumor events such as increased apoptosis and decreased angiogenesis; and also presented pro-tumorigenic events; most notably was the increased fibrosis and metastasis to the liver. These observations have shed light on the possible role the CXCR2 on the other TME component especially pancreatic stellate cells (PSCs). The aim of this study is to determine the differential effect of K-ras status and CXCR2 chemokines expression in the PC cells on their interaction with PSCs. Unidirectional segregated co-culture studies were conducted using conditioned media (CM). CM collected from PSCs were used to grow multiple PC cell lines, and CM obtained from different PC cell lines were used to culture PSCs. Cellular proliferation and gene expression were analyzed. Furthermore, PSCs proliferation potentials were assessed as those cells treated with exogenous CXCL1 and CXCL8, or PC cell lines derived-CM in the presence or absence of CXCR2 antagonists. Co-culture studies revealed that PSC-derived CM had increased proliferation of PC cell lines positive to oncogenic K-ras as well as increasing their expression of multiple chemokines such as CXCL1, CXCL5, and CCL2. On the other hand, PC cell lines with a wildtype K-ras were inhibited by PSC-derived CM treatment. PSCs proliferation potentials were decreased with CM derived from oncogenic K-ras positive PC cell lines and increased with CM derived from PC cell lines with wildtype K-ras. Furthermore, CM from oncogenic K-ras PC cell lines has increased the expression of multiple pro-tumorigenic genes in the PSCs including cytokines such as IL-10, IL-4 and IL-13, and chemokines such as CXCL2 and CXCL7. Also, treating PSCs with exogenous CXCL1 and CXCL8 exhibited a similar proliferation inhibition to that observed with oncogenic K-ras PC derived CM treatment. CXCR2 antagonists have shown rescued inhibition or enhanced proliferation of PSCs when incorporated with CXCR2 chemokine or PC cell line derived CM treatment. These observations suggest that K-ras status of PC dictates their interaction with PSCs within the TME. We have demonstrated that oncogenic K-ras through increased production of CXCR2 chemokine would dampen PSCs proliferation and further orient them to support tumor progression by increased expression of pro-tumorigenic genes. Besides, we observed that CXCR2 inhibition in PSCs would increase their proliferation which may further their fibrogenic activity. Citation Format: Mohammad Awaji, Michelle Varney, Abhilasha Purohit, Surinder K. Batra, Rakesh K. Singh. CXCR2 acts as a checkpoint regulator of the pancreatic stellate cells activity within pancreatic cancer tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 942. doi:10.1158/1538-7445.AM2017-942

Abstract 1135: DRD2 is critical for pancreatic cancer and promises pharmacological therapy by already established antagonists

Abstract Introduction and aims: Although the overall five-year survival of all patients with cancer stands at 63%, for pancreatic cancer patients, it is a disheartening 8% - a number that remains largely unchanged for three decades. Of the patients diagnosed with pancreatic cancer, about 85% exhibit pancreatic ductal adenocarcinoma (PDAC). Most of these patients die within 4 to 6 months after diagnosis. The poor prognosis is caused by the detection at only late stages, and lack of effective options for chemotherapy. The widely used chemotherapeutic agent gemcitabine, confers a median survival advantage of only 6 months, and resistance to therapy develops in the vast majority of patients. Given this poor prognosis of patients with PDAC, there is an urgent need to find more effective therapies. Experimental procedures: Microarrays were used to perform global gene expression profiling in 195 PDAC and 41 normal pancreatic tissue samples. Using these profiling data, we undertook an extensive analysis of PDAC transcriptome by superimposing the pathway context and interaction networks of aberrantly expressed genes to identify factors with central roles in PDAC pathways. Next, tissue microarray analysis (TMA) were used to verify the expression of the candidate target in independent set of 152 samples comprising 40 normal pancreatic tissues, 49 chronic pancreatitis sections (CP) and 63 PDAC samples. We further validated the functional relevance of the candidate molecule through RNA interference (RNAi) and pharmacological inhibition in vitro and in vivo. Results: We identified dopamine receptor D2 (DRD2) as a key modulator of cancer pathways in PDAC. DRD2 up-regulation at the protein level was validated in a large independent sample cohort. Most importantly, we found that blockade of DRD2, through RNAi or pharmacological inhibition using FDA-approved antagonists hampers the proliferative and invasive capacities of pancreatic cancer cells while modulating cAMP and endoplasmic reticulum stress pathways. Also, we observed a potent effect of DRD2 antagonists on inhibition of cancer cell proliferation using different model of primary and metastatic tumor cells derived from spontaneous pancreatic cancer mouse models and patient-derived pancreatic adenocarcinoma mouse xenograft (PDX) models. Conclusions: Our findings demonstrate that inhibiting DRD2 represents a novel therapeutic approach for PDAC. Since DRD2 inhibitors are already in the clinic for the management of schizophrenia, our results from this study could support a drug repurposing strategy to expedite clinical evaluation of these agents as novel therapy against pancreatic cancer. Citation Format: Pouria Jandaghi, Hamed S. Najafabadi, Andrea Bauer, Andreas I. Papadakis, Matteo Fassan, Anita Hall, Anie Monast, Maryam Safisamghabadi, Magnus von Knebel Doeberitz, John P. Neoptolemos, Eithne Costello, William Greenhalf, Aldo Scarpa, Bence Sipos, Daniel Auld, Mark Lathrop, Morag Park, Markus W. Büchler, Oliver Strobel, Thilo Hackert, Nathalia Giese, George Zogopoulos, Veena Sangwan, Sidong Huang, Jörg D. Hoheisel, Yaser Riazalhosseini. DRD2 is critical for pancreatic cancer and promises pharmacological therapy by already established antagonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1135. doi:10.1158/1538-7445.AM2017-1135

Abstract 1867: Near infrared fluorescent imaging of brain tumor with IR780 dye incorporated phospholipid nanoparticles

Abstract Background: Near-IR fluorescence (NIRF) imaging is becoming a promising approach in preclinical tumor detection and clinical image-guided oncological surgery. While heptamethine cyanine dye IR780 has excellent tumor targeting and imaging potential, its hydrophobic properties limit its clinical use. In this study, we developed nanoparticle formulations to facilitate the use of IR780 for fluorescent imaging of malignant brain tumor. Methods: Self-assembled IR780-liposomes and IR780-phospholipid micelles were prepared and their NIRF properties were characterized. The intracellular accumulation of IR780-nanoparticles in glioma cells were determined using confocal microscopy. The in vivo brain tumor targeting and NIRF imaging capacity of IR780-nanoparticles were evaluated using U87MG glioma ectopic and orthotopic xenograft models and a spontaneous glioma mouse model driven by RAS/RTK activation. Results: The loading of IR780 into liposomes or phospholipid micelles was efficient. The particle diameter of IR780-liposomes and IR780-phospholipid micelles were 95 nm and 26 nm, respectively. While stock solutions of each preparation were maintained at ready-to-use condition, the IR780-phospholipid micelles were more stable. In tissue culture cells, IR780-nanoparticles prepared by either method accumulated in mitochondria, however, in animals the IR780-phospholipid micelles showed enhanced intra-tumoral accumulation in U87MG ectopic tumors. Moreover, IR780-phospholipid micelles also showed preferred intracranial tumor accumulation and potent NIRF signal intensity in glioma orthotopic models at a real-time, non-invasive manner. Conclusion: The IR780-phospholipid micelles demonstrated tumor-specific NIRF imaging capacity in glioma preclinical mouse models, providing great potential for clinical imaging and image-guided surgery of brain tumors. Citation Format: Shihong Li, Jennifer Johnson, Anderson Peck, Qian Xie. Near infrared fluorescent imaging of brain tumor with IR780 dye incorporated phospholipid nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1867. doi:10.1158/1538-7445.AM2017-1867

Abstract 3678: Muc4 nanovaccine and checkpoint blockade based combination immunotherapy for pancreatic cancer

Abstract Background: Pancreatic cancer (PC) is a highly metastatic and therapy-resistant malignancy characterized by immunosuppressive tumor microenvironment (TME) mucin overexpression. MUC4 is most differentially overexpressed mucin and functionally contributes to disease aggressiveness and is thus ideal candidate for targeted therapies. Tumor cells are also known to express programmed death ligand 1 (PD-L1) to suppress effector immune cells activity via PD-L1-PD-1 axis. We developed a novel vaccine using recombinant MUC4 fragments and exploiting adjuvant-like properties of amphiphilic polyanhydride-based nanoparticle delivery system. The strong involvement of MUC4 in disease aggressiveness and PD-L1 in immunosuppression makes a compelling case for their combined targeting. We hypothesize that combined overexpression of MUC4 and PD-L1 expression in pancreatic tumor microenvironment contributes to immunosuppressive and aggressive tumor behavior. Methods: Recombinant MUC4β fragment was encapsulated into polyanhydride nanoparticles (20:80 CPTEG:CPH) via nanoprecipitation. Murine PC cell lines (KCT960) derived from spontaneous pancreatic cancer mouse model (KPC mice) were transfected with human MUC4 expressing construct known as KCT960-mini-MUC4 cells for in-vivo studies. Flow cytometry, immunoblotting, PCR and immunofluorescence techniques were utilized to perform to characterize the immune response and target expression. Results: MUC4β nanovaccine immunized mice exhibited slower tumor growth kinetics than unimmunized control mice. We investigated tumor infiltrating lymphocytes (TILs) and necrosis in the tumor bed. We observed a positive correlation between TILs and tumor regression. Accumulation of infiltrating CD8+ and CD4+ T cells was greater in mice receiving the MUC4-Nanovaccine compared to soluble MUC4 delivered with blank nanoparticles, indicating the benefit of sustained availability of antigen via encapsulation. However, we did not observe complete tumor regression and ex vivo studies indicated induction of IFN-gamma by MUC4 vaccine. Based on this, we rationalized that PD-L1 expression by MUC4-expressing tumor cells suppressed and inhibited the therapeutic benefits of the nanovaccine in-vivo. We observed differential surface expression of PD-L1 on endogenously MUC4 expressing and negative PC cell lines. Paired analysis of MUC4 CRISPR knockdown and scrambled Capan-1 cell lines further suggested similar positive correlation. Conclusion: MUC4 nanovaccine suppressed tumor progression in-vivo indicating its potential for immunotherapy of PC. Positive correlation observed between MUC4 and PD-L1 in PC cell lines suggests an underlying molecular mechanism for establishing an immunosuppressive and aggressive PC tumor microenvironment and provides a strong rationale for evaluating MUC4 vaccine in combination with immune checkpoint blockade agents. Citation Format: Kasturi Banerjee, Shailendra Gautam, Waseem Mohd. Nasser, Prakash Kshirsagar, Kathleen Ross, Gaelle Spagnol, Abhijit Aithal, Paul Sorgen, Surinder Kumar Batra, Sushil Kumar, Balaji Narasimhan, Maneesh Jain. Muc4 nanovaccine and checkpoint blockade based combination immunotherapy for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3678. doi:10.1158/1538-7445.AM2017-3678

Abstract 1258: Lifestyle improvements delay cancer onset and progression by adapting endocrine and immune signaling in mouse models

Abstract Metabolic syndromes instigate substantial morbidity and mortality worldwide via its negative effects of many diseases including cancer. Obesity in particular has been deemed a causative factor for breast cancer in post-menopausal women, while worsening prognosis regardless of menopausal status. Importantly, progression of many metabolic syndromes can largely be slowed or reversed with aggressive lifestyle changes, thus also having the potential to mitigate cancer onset. A mouse model of environmental enrichment (EE) to improve motosensory, cognitive, and social stimulation by increasing physical engagement and social interaction triggers vast improvements in overall health. These include boosting mental health, reducing adiposity, prevention of diet-induced obesity (DIO), promoting the white to brown fat transition, enhancing insulin sensitivity, improving immune function, limiting inflammation, and inhibition of cancer growth. We have elucidated the central mechanism of EE to be the activation of the hypothalamic-sympathoneural-adipocyte (HSA) axis, a specific neuroendocrine route in which the brain communicates with adipose tissue via the sympathetic nervous system. Once activated, norepinephrine (NE) is released directly onto adipose tissue which induces the observed metabolic improvements. Breast cancer is uniquely exposed to this adipose-NE so we sought to explore this distinctive microenvironment in the context of both local and systemic endocrine and immune signaling in varied body mass states and menopausal statuses. Our data showed that EE diminished leptin levels and delayed cancer onset in the MMTV-PyMT spontaneous mouse model of breast cancer and slowed growth in a DIO allograft model. In contrast, EE failed to attenuate tumor progression in ob/ob mice in the absence of leptin, but rather enhanced tumor growth, suggesting that the leptin-NE relationship is one of the key peripheral mediators of the EE anti-breast cancer effects. We also observed significant immune cell modulation in the adipose tissue following EE. Initial characterization revealed a decrease in the proportion of tumor-associated macrophages (TAMs) and an overall anti-inflammatory response which together with the drop in leptin may contribute to the observed anti-cancer effects. We plan to further elucidate this EE-induced adipose and immune cell modulation with the ultimate goal of developing therapeutic strategies to treat both metabolic syndromes as well as associated and possibly independent cancer types. Citation Format: Grant D. Foglesong, Stephen M. Bergin, Wei Huang, Michael A. Caligiuri, Lei Cao. Lifestyle improvements delay cancer onset and progression by adapting endocrine and immune signaling in mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1258. doi:10.1158/1538-7445.AM2017-1258

Spontaneous loss of B lineage transcription factors leads to pre-B leukemia in Ebf1+/\u2013Bcl-xLTg mice

Early B-cell factor 1 (EBF1) plays a central role in B-cell lineage specification and commitment. Loss of this critical transcription factor is strongly associated with high-risk, relapsed and therapy-resistant B–cell-acute lymphoblastic leukemia, especially in children. However, Ebf1 haploinsufficient mice exhibit a normal lifespan. To determine whether prolonged survival of B cells would enable tumorigenesis in Ebf1 haploinsufficient animals, we generated Ebf1+/–Bcl-xLTg mice, which express the anti-apoptotic factor Bcl-xL in B cells. Approximately half of Ebf1+/–Bcl-xLTg mice develop aggressive oligoclonal leukemia as they age, which engrafts in congenic wild-type recipients without prior conditioning. The neoplastic cells display a pre-B phenotype and express early developmental- and natural killer cell/myeloid-markers inappropriately. In addition, we found tumor cell-specific loss of several transcription factors critical for maintaining differentiation: EBF1, TCF3 and RUNX1. However, in the majority of tumors, loss of Ebf1 expression was not due to loss of heterozygosity. This is the first spontaneous mouse model of pre-B leukemia to demonstrate inappropriate expression of non-B-cell-specific genes associated with loss of Ebf1, Tcf3 and Runx1 expression.

Abstract 1978: Identification of the metastatic cell populations in a spontaneous mouse model of melanoma

Abstract Melanoma is the deadliest form of skin cancer due to its high propensity to metastasize and resistance to current therapies. We have created a spontaneous mouse model of metastatic melanoma (Dct-Grm1/K5-Edn3) where metastasis to the lungs is 80% penetrant. The primary tumors of these mice present cellular heterogeneity with cells at varying levels of differentiation. The main goal of this study is to determine the metastatic potential of the primary tumor resident Tyrosinase positive cells and evaluate the dynamic pattern of gene expression as those cells move from the primary tumors to the sites of metastasis. To accomplish this aim we crossed the Dct-Grm1/K5-Edn3 mice to CreERT2/ ROSAmT/mG mice to indelibly label Tyrosinase cell populations within the primary tumor by topical application of 4-hydroxytamoxifen at the tumor site. In vivo lineage tracing and characterization of those labeled cells was performed in the metastatic lesions. We found that Tyrosinase positive cells enter the circulation before the appearance of any noticeable tumor or nevus and establish close interactions with the vasculature as single cells. Metastatic cells in close association with the inner wall of the vasculature lose pigmentation and do not express melanocytic markers. Interestingly, those intravascular cells mimic endothelial cell properties with the expression of platelet endothelial cell adhesion molecule (PECAM-1, also known as CD31). In the lung tissue, the primary tumor derived Tyrosinase positive cells or their progeny can survive and establish successful metastases with pigmentation. This in vivo lineage tracing system in mouse will be a powerful model to evaluate and help us understand the etiology and pathogenesis of melanoma metastasis. Further characterization of those more aggressive cells in melanoma will allow for the development of new prognostic tests and novel therapeutic strategies to eliminate metastasis. Citation Format: Xiaoshuang Li, Raul Torres, Lidia Kos. Identification of the metastatic cell populations in a spontaneous mouse model of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1978. doi:10.1158/1538-7445.AM2017-1978

Effects of phosphodiesterase 3A modulation on murine cerebral microhemorrhages

BackgroundCerebral microbleeds (CMB) are MRI-demonstrable cerebral microhemorrhages (CMH) which commonly coexist with ischemic stroke. This creates a challenging therapeutic milieu, and a strategy that simultaneously protects the vessel wall and provides anti-thrombotic activity is an attractive potential approach. Phosphodiesterase 3A (PDE3A) inhibition is known to provide cerebral vessel wall protection combined with anti-thrombotic effects. As an initial step in the development of a therapy that simultaneously treats CMB and ischemic stroke, we hypothesized that inhibition of the PDE3A pathway is protective against CMH development.MethodsThe effect of PDE3A pathway inhibition was studied in the inflammation-induced and cerebral amyloid angiopathy (CAA)-associated mouse models of CMH. The PDE3A pathway was modulated using two approaches: genetic deletion of PDE3A and pharmacological inhibition of PDE3A by cilostazol. The effects of PDE3A pathway modulation on H&E- and Prussian blue (PB)-positive CMH development, BBB function (IgG, claudin-5, and fibrinogen), and neuroinflammation (ICAM-1, Iba-1, and GFAP) were investigated.ResultsRobust development of CMH in the inflammation-induced and CAA-associated spontaneous mouse models was observed. Inflammation-induced CMH were associated with markers of BBB dysfunction and inflammation, and CAA-associated spontaneous CMH were associated primarily with markers of neuroinflammation. Genetic deletion of the PDE3A gene did not alter BBB function, microglial activation, or CMH development, but significantly reduced endothelial and astrocyte activation in the inflammation-induced CMH mouse model. In the CAA-associated CMH mouse model, PDE3A modulation via pharmacological inhibition by cilostazol did not alter BBB function, neuroinflammation, or CMH development.ConclusionsModulation of the PDE3A pathway, either by genetic deletion or pharmacological inhibition, does not alter CMH development in an inflammation-induced or in a CAA-associated mouse model of CMH. The role of microglial activation and BBB injury in CMH development warrants further investigation.

Targeting innate immunity to downmodulate adaptive immunity and reverse type 1 diabetes.

Type 1 diabetes (T1D) is characterized by specific destruction of pancreatic insulin-producing beta cells accompanied by evidence of beta-cell-directed autoimmunity such as autoreactive T cells and islet autoantibodies (IAAs). Currently, T1D cannot be prevented or reversed in humans. T1D is easy to prevent in the nonobese diabetic (NOD) spontaneous mouse model but reversing new-onset T1D in mice is more difficult. Since the discovery of the T-cell receptor in the 1980s and the subsequent identification of autoreactive T cells directed toward beta-cell antigens (eg, insulin, glutamic acid decarboxylase), the dream of antigen-specific immunotherapy has dominated the field with its promise of specificity and limited side effects. While such approaches have worked in the NOD mouse, however, dozens of human trials have failed. Broader immunosuppressive approaches (originally cyclosporine, subsequently anti-CD3 antibody) have shown partial successes (e.g., prolonged C peptide preservation) but no major therapeutic efficacy or disease reversal. Human prevention trials have failed, despite the ease of such approaches in the NOD mouse. In the past 50 years, the incidence of T1D has increased dramatically, and one explanation is the “hygiene hypothesis”, which suggests that decreased exposure of the innate immune system to environmental immune stimulants (e.g., bacterial products such as Toll-like receptor (TLR) 4-stimulating lipopolysaccharide [LPS]) dramatically affects the adaptive immune system and increases subsequent autoimmunity. We have tested the role of innate immunity in autoimmune T1D by treating acute-onset T1D in NOD mice with anti-TLR4/MD-2 agonistic antibodies and have shown a high rate of disease reversal. The TLR4 antibodies do not directly stimulate T cells but induce tolerogenic antigen-presenting cells (APCs) that mediate decreased adaptive T-cell responses. Here, we review our current knowledge and suggest future prospects for targeting innate immunity in T1D immunotherapy.

The expression profiles and regulation of PD-L1 in tumor-induced myeloid-derived suppressor cells

Abstract Programmed death-ligand 1 (PD-L1) is an inhibitory ligand that binds to PD-1 to suppress T cell activation. PD-L1 is constitutively expressed and inducible in tumor cells, but the expression profiles and regulatory mechanism of PD-L1 in myeloid-derived suppressor cells (MDSCs) are largely unknown. We report that PD-L1 is abundantly expressed in tumor-infiltrating immune cells in human colon carcinoma, and 25–67% CD11b+CD33+ myeloid-derived suppressor cells (MDSCs) from peripheral blood of human colon cancer patients are PD-L1+. Using a spontaneous colon cancer mouse model, we observed that PD-L1+MDSCs increased significantly in tumor-bearing mice as compared to MDSCs from tumor-free mice. Interestingly, the highest PD-L1+ MDSCs were observed in the tumor microenvironment in which 56–71% tumor-infiltrating MDSCs are PD-L1+ in vivo. In contrast, PD-L1+ MDSCs are significantly less in secondary lymphoid organs and peripheral blood as compared to the tumor tissues, whereas, bone marrow MDSCs are essentially PD-L1− in tumor-bearing mice. IFNg is highly expressed in cells of the tumor tissues and IFNg neutralization significantly decreased PD-L1+ MDSCs in the tumor microenvironment in vivo. However, IFNg-activated pSTAT1 does not bind to the cd274 promoter in CD11b+Gr1+PD-L1+ myeloid cells. Instead, pSTAT1 activates expression of IRF1, IRF5, IRF7 and IRF8 in MDSCs, and only pSTAT1-activated IRF1 binds to an unique IRF-binding sequence element in vitro and chromatin in vivo in the cd274 promoter to activate PD-L1 transcription. Our data determine that PD-L1 is highly expressed in tumor-infiltrating MDSCs and in a less degree in lymphoid organs, and the pSTAT1-IRF1 axis regulates PD-L1 expression in MDSCs.