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Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer with very few treatment options. Less than 10 percent of patients diagnosed with PDAC survive 5 years post diagnosis. Mutations in CDKN2A, SMAD4, KRAS and P53 have been well linked to the development of PDAC. Preclinical murine models have been developed that leverage key driver mutations and have significantly contributed to our understanding of PDAC. One such genetically engineered mouse model (GEMM) that has emerged as an important tool in PDAC investigations is the KPC mouse (LSL-KrasG12D/+LSL-Trp53R172H/+Pdx-1-Cre) that spontaneously develops pancreatic tumors at ~14-16 weeks of age. Cadherin-11 (Cdh11), a cell-to-cell adhesion molecule, is highly expressed in desmoplastic stroma, a characteristic of PDAC, that leads to difficulties in drug accessibility and has been hypothesized to contribute to chemotherapeutic resistance. However, the mechanisms by which Cdh11 deficiency in the stromal microenvironment of PDAC-bearing mice (KPC) influences therapeutic outcomes, has yet to be fully understood. Single-cell RNA sequencing (scRNAseq) of both the non-immune (CD45-) and immune (CD45+) cellular compartments of tumor bearing (KPC/Cdh11+/+), tumor bearing Cdh11 deficient (KPC/Cdh11+/-), non-tumor bearing Cdh11 deficient (Cdh11-/+) and wildtype (KP) were performed. We observed changes in the abundance and types of infiltrating immune cells (T-cells, B-cells, myeloid lineage cells) of KPC/Cdh11+/- tumors when compared to tumors harvested from KPC/Cdh11+/+ mice. KPC/Cdh11+/+ pancreata had significantly more myeloid cells while KPC/Cdh11+/- tumors favored an increase in the numbers of infiltrating B- and T- cells. Genes upregulated in infiltrating T-cells specific to KPC/Cdh11+/+ mice include Spp1, Ifi30, Apoe, Ifitm3, Fn1. The increase in B and T cell infiltration was specific to the Cdh11-/+ deficient background, since both pancreata from KPC/Cdh11+/- and Cdh11-/+ mice had elevated levels of infiltration, compared to the KPC group. We also observed a decrease in the number of antigen-presenting cancer associated fibroblasts (apCAFs) in Cdh11-/+ and KPC/Cdh11+/- pancreata, denoted by the lack of CD74+ fibroblasts. Further validation of these findings will help to define the role of Cdh11-/+ in modulating B and T-cell behavior in addition to providing insight into Cdh11-/+ as a therapeutic target for PDAC through altering the tumor microenvironment. Citation Format: Kelly A. Martin, Aimy Sebastian, Nicholas Hum, Ivana Peran, Stephen Byers, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela Loots. Loss of Cadherin-11 in PDAC induces altered immune cell infiltration and remodels stromal landscape [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 2741.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with limited and, very often, ineffective medical and surgical therapeutic options. The treatment of patients with advanced unresectable PDAC is restricted to systemic chemotherapy, a therapeutic intervention to which most eventually develop resistance. Recently, nab-paclitaxel (n-PTX) has been added to the arsenal of first-line therapies, and the combination of gemcitabine and n-PTX has modestly prolonged median overall survival. However, patients almost invariably succumb to the disease, and little is known about the mechanisms underlying n-PTX resistance. Using the conditionally reprogrammed (CR) cell approach, we established and verified continuously growing cell cultures from treatment-naïve patients with PDAC. To study the mechanisms of primary drug resistance, nab-paclitaxel–resistant (n-PTX-R) cells were generated from primary cultures and drug resistance was verified in vivo, both in zebrafish and in athymic nude mouse xenograft models. Molecular analyses identified the sustained induction of c-MYC in the n-PTX-R cells. Depletion of c-MYC restored n-PTX sensitivity, as did treatment with either the MEK inhibitor, trametinib, or a small-molecule activator of protein phosphatase 2a. Implications: The strategies we have devised, including the patient-derived primary cells and the unique, drug-resistant isogenic cells, are rapid and easily applied in vitro and in vivo platforms to better understand the mechanisms of drug resistance and for defining effective therapeutic options on a patient by patient basis.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinomas (PDAC) are characterized by fibrosis and an abundance of cancer-associated fibroblasts (CAFs). We investigated strategies to disrupt interactions among CAFs, the immune system, and cancer cells, focusing on adhesion molecule cadherin 11 (CDH11), which has been associated with other fibrotic disorders and is expressed by activated fibroblasts. METHODS: We compared levels of CDH11 mRNA in human pancreatitis and pancreatic cancer tissues and cells, compared with normal pancreas, and measured levels of CDH11 protein in human and mouse pancreatic lesions and normal tissues. We crossed p48-Cre;LSL-Kras(G12D/+);LSL-Trp53(R172H/+) (KPC) mice with CDH11-knockout mice and measured survival times of offspring. Pancreata were collected and analyzed by histology, immunohistochemistry, and (single-cell) RNA sequencing; RNA and proteins were identified by imaging mass cytometry. Some mice were given injections of PD1 antibody or gemcitabine and survival was monitored. Pancreatic cancer cells from KPC mice were subcutaneously injected into Cdh11(+/+) and Cdh11(−/−) mice and tumor growth was monitored. Pancreatic cancer cells (mT3) from KPC mice (C57BL/6), were subcutaneously injected into Cdh11(+/+) (C57BL/6J) mice and mice were given injections of antibody against CDH11, gemcitabine, or small molecule inhibitor of CDH11 (SD133) and tumor growth was monitored. RESULTS: Levels of CDH11 mRNA and protein were significantly higher in CAFs than in pancreatic cancer epithelial cells, human or mouse pancreatic cancer cell lines, or immune cells. KPC/Cdh11(+/−) and KPC/Cdh11(−/−) mice survived significantly longer than KPC/Cdh11(+/+) mice. Markers of stromal activation entirely surrounded pancreatic intraepithelial neoplasias in KPC/Cdh11(+/+) mice and incompletely in KPC/Cdh11(+/−) and KPC/Cdh11(−/−) mice, whose lesions also contained fewer FOXP3(+) cells in the tumor center. Compared with pancreatic tumors in KPC/Cdh11(+/+) mice, tumors of KPC/Cdh11(+/−) mice had increased markers of antigen processing and presentation; more lymphocytes and associated cytokines; decreased extracellular matrix components; and reductions in markers and cytokines associated with immunosuppression. Administration of the PD1 antibody did not prolong survival of KPC mice with 0, 1, or 2 alleles of Cdh11. Gemcitabine extended survival only of KPC/Cdh11(+/−) and KPC/Cdh11(−/−) mice or reduced subcutaneous tumor growth in mT3 engrafted Cdh11(+/+) mice given in combination with the CDH11 antibody. A small molecule inhibitor of CDH11 reduced growth of pre-established mT3 subcutaneous tumors only if T and B cells were present in mice. CONCLUSIONS: Knockout or inhibition of CDH11, which is expressed by CAFs in the pancreatic tumor stroma, reduces growth of pancreatic tumors, increases their response to gemcitabine, and significantly extends survival of mice. CDH11 promotes immunosuppression and extracellular matrix deposition, and might be developed as a therapeutic target for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most incurable types of cancer. Cancer that develops in the acinar cells of the pancreas is typically not diagnosed until later stages, such as stage 3 or 4. As such, this form of cancer is particularly lethal with only 9% of patients reaching 5-year survival. PDAC is known to have a particularly dense extracellular matrix composed of fibroblasts, which have been previously shown to play an important role in promoting resistance to drug therapy. Characterization of the stromal networks involved in PDAC tumor development as well as protein markers of fibroblast subpopulations within the tumor stroma are critical to developing new fibroblast-targeted therapeutic approaches as well as understanding key signaling molecules that ultimately promote tumor progression and drug resistance. Single-cell RNA sequencing (scRNAseq) was utilized to analyze cancer-associated fibroblasts (CAFs) from KPC mouse-derived, MT3 subcutaneous murine allografts along with two fibroblast lines derived from human PDAC tumors: CRC-811 and IA-1340. Single cell gene expression profiling and subsequent analysis of MT3-derived CAFs resulted in the identification of three CAF subpopulations including a myofibroblast subpopulation that expressed high levels of smooth muscle actin (Acta2), which was also observed in both human samples. Fibroblast subpopulations enriched in Acta2 expression, expressed high levels of Wnt5a along with several other secreted factors including Tgfb1, Tgfb2 and Ctgf. Wnt5a is a secreted protein that activates non-canonical Wnt signaling pathways and is known to regulate normal developmental processes, including proliferation, differentiation, migration, adhesion and polarity. However Wnt5a is not natively expressed in MT3 cancer cells derived from syngeneic tumors, but potentially in the stroma. We hypothesize it is exclusively derived from fibroblasts. Previously it has been shown that Wnt5a inhibition suppressed gastric cancer metastasis, therefore further validation of the role of myofibroblast-derived Wnt5a on PDAC disease progression is warranted. This study received funding by LDRD 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). IM Release Number: LLNL-ABS-798442. Citation Format: Kelly A. Martin, Aimy Sebastian, Nicholas R. Hum, Stephen Byers, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. Analysis of stromal myofibroblasts identifies secreted proteins that promote pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2757.

Anterior cruciate ligament (ACL) injuries often result in post-traumatic osteoarthritis (PTOA). To better understand the molecular mechanisms behind PTOA development following ACL injury, we profiled ACL injury-induced transcriptional changes in knee joints of three mouse strains with varying susceptibility to OA: STR/ort (highly susceptible), C57BL/6J (moderately susceptible) and super-healer MRL/MpJ (not susceptible). Right knee joints of the mice were injured using a non-invasive tibial compression injury model and global gene expression was quantified before and at 1-day, 1-week, and 2-weeks post-injury using RNA-seq. Following injury, injured and uninjured joints of STR/ort and injured C57BL/6J joints displayed significant cartilage degeneration while MRL/MpJ had little cartilage damage. Gene expression analysis suggested that prolonged inflammation and elevated catabolic activity in STR/ort injured joints, compared to the other two strains may be responsible for the severe PTOA phenotype observed in this strain. MRL/MpJ had the lowest expression values for several inflammatory cytokines and catabolic enzymes activated in response to ACL injury. Furthermore, we identified several genes highly expressed in MRL/MpJ compared to the other two strains including B4galnt2 and Tpsab1 which may contribute to enhanced healing in the MRL/MpJ. Overall, this study has increased our knowledge of early molecular changes associated with PTOA development.

Whole genome gene expression datasets of tissues or homogeneous cell populations isolated from healthy or disease states (humans or animals) provide a glimpse of the entire transcriptome within a narrow temporal and spatial functional window and thereby assist the identification of key molecules and transcriptional regulatory networks within a biological system. Clusters of coexpressed genes can guide the discovery of candidate lineage markers and regulators of cell function. While gene expression profiling using microarrays and RNAseq have not yet been explored to their full potential in the field of skeletal biology, work over the last decade has presented data that have greatly benefited from genomic approaches. Here we will review recent work that has used gene expression profiling as a tool for furthering our understanding of: (1) cell specific gene expression and bone metabolism; (2) age-dependent and anatomical location-dependent differences in skeletal gene expression; (3) biomechanical effects on bone; (4) molecular changes in animal models of bone disease; (5) patients with osteoporosis (OP), and (6) noncoding RNAs in bone development and disease. We will focus primarily on literature that describes profiling of homogenous cells and tissue microdissection from patients or live animals, to provide an overview of genomic profiling that may more faithfully adhere to in vivo bone physiology.

Dynamic interaction between prostate cancer and the bone microenvironment is a major contributor to metastasis of prostate cancer to bone. In this study, we utilized an in vitro co-culture model of PC3 prostate cancer cells and osteoblasts followed by microarray based gene expression profiling to identify previously unrecognized prostate cancer-bone microenvironment interactions. Factors secreted by PC3 cells resulted in the up-regulation of many genes in osteoblasts associated with bone metabolism and cancer metastasis, including Mmp13, Il-6 and Tgfb2, and down-regulation of Wnt inhibitor Sost. To determine whether altered Sost expression in the bone microenvironment has an effect on prostate cancer metastasis, we co-cultured PC3 cells with Sost knockout (Sost(KO)) osteoblasts and wildtype (WT) osteoblasts and identified several genes differentially regulated between PC3-Sost(KO) osteoblast co-cultures and PC3-WT osteoblast co-cultures. Co-culturing PC3 cells with WT osteoblasts up-regulated cancer-associated long noncoding RNA (lncRNA) MALAT1 in PC3 cells. MALAT1 expression was further enhanced when PC3 cells were co-cultured with Sost(KO) osteoblasts and treatment with recombinant Sost down-regulated MALAT1 expression in these cells. Our results suggest that reduced Sost expression in the tumor microenvironment may promote bone metastasis by up-regulating MALAT1 in prostate cancer.

Patients with anterior cruciate ligament (ACL) rupture are two times as likely to develop posttraumatic osteoarthritis (PTOA). Annually, there are ∼900,000 knee injuries in the United States, which account for ∼12% of all osteoarthritis (OA) cases. PTOA leads to reduced physical activity, deconditioning of the musculoskeletal system, and in severe cases requires joint replacement to restore function. Therefore, treatments that would prevent cartilage degradation post‐injury would provide attractive alternatives to surgery. Sclerostin (Sost), a Wnt antagonist and a potent negative regulator of bone formation, has recently been implicated in regulating chondrocyte function in OA. To determine whether elevated levels of Sost play a protective role in PTOA, we examined the progression of OA using a noninvasive tibial compression overload model in SOST transgenic (SOSTTG) and knockout (Sost‐/‐) mice. Here we report that SOSTTG mice develop moderate OA and display significantly less advanced PTOA phenotype at 16 weeks post‐injury compared with wild‐type (WT) controls and Sost‐/‐. In addition, SOSTTG built ∼50% and ∼65% less osteophyte volume than WT and Sost‐/‐, respectively. Quantification of metalloproteinase (MMP) activity showed that SOSTTG had ∼2‐fold less MMP activation than WT or Sost‐/‐, and this was supported by a significant reduction in MMP2/3 protein levels, suggesting that elevated levels of SOST inhibit the activity of proteolytic enzymes known to degrade articular cartilage matrix. Furthermore, intra‐articular administration of recombinant Sost protein, immediately post‐injury, also significantly decreased MMP activity levels relative to PBS‐treated controls, and Sost activation in response to injury was TNFα and NF‐κB dependent. These results provide in vivo evidence that sclerostin functions as a protective molecule immediately after joint injury to prevent cartilage degradation. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Bladder cancer is among the top ten most common cancers, with about ~380,000 new cases and ~150,000 deaths per year worldwide. Tumor relapse following chemotherapy treatment has long been a significant challenge towards completely curing cancer. We have utilized a patient-derived bladder cancer xenograft (PDX) platform to characterize molecular mechanisms that contribute to relapse following drug treatment in advanced bladder cancer. Transcriptomic profiling of bladder cancer xenograft tumors by RNA-sequencing analysis, before and after relapse, following a 21-day cisplatin/gemcitabine drug treatment regimen identified methionine adenosyltransferase 1a (MAT1A) as one of the significantly upregulated genes following drug treatment. Survey of patient tumor sections confirmed elevated levels of MAT1A in individuals who received chemotherapy. Overexpression of MAT1A in 5637 bladder cancer cells increased tolerance to gemcitabine and stalled cell proliferation rates, suggesting MAT1A upregulation as a potential mechanism by which bladder cancer cells persist in a quiescent state to evade chemotherapy.

Exosomes are endosomal secreted vesicles containing a variety of genetic and non-genetic material that can be transferred between cells (DNA, RNA, protein, lipid, etc.). Cancer derived exosomes have been implicated in a wide range of cancer mechanisms such as promotion of tumor growth, tumor angiogenesis, immune evasion, drug resistance, and metastasis. In addition to functional significance, cancer exosomes may possess novel biomarkers with potential uses for non-invasive liquid biopsies for cancer detection and monitoring of disease progression. Here we examined differences that may exist in extracellular vesicles or exosomes (EVs) shed by cancer cell lines with various metastatic potential (MDA-MB-231, highly metastatic; MCF-7, weakly metastatic; MCF-10a, normal breast epithelial cells). Morphological analysis of EVs was performed using fluorescent Nanosight Tracking Analysis and revealed decreased mean diameter of EVs derived from MCF-7 cells compared to the other two cell lines, there was also increased exosome particle yield in this cell line. Small RNAs from EVs cargo were also analyzed via sequencing (exoRNA-Seq). The majority of sequenced RNA aligned to coding regions of the human genome (~60%) across all cell lines. Non-coding RNA classification also showed little variability across cell lines examined regardless of metastatic potential, with ~38% of RNA reads corresponding to non-coding RNAs. Pairwise comparison of these cell lines demonstrated that each line packaged unique cargos into their shed exosomes. The non-coding regions corresponded to a variety of small RNAs (miRNA, snoRNA, snRNA, miscRNA, rRNA) but included other RNA features such as pseudogenes and antisense RNAs. We found 304 genes significantly up-regulated and 150 genes significantly down-regulated when comparing metastatic cancer exosomes (MCF-7 and MDA-MB-231) to normal, non-tumorigenic exosomes (MCF-10A). Among examined microRNAs we found several miRNAs associated with metastatic behavior that have previously been implicated in cancer biology, which will be prioritized for validation. Also, 21 RNAs were upregulated at high levels in exosomes shed by metastatic cells, and their expression level was directly proportional with the metastatic character, suggesting that these RNAs may be tested for their potential as biomarkers. Future research will expand upon these newly identified genetic signatures of metastatic exosomal cargo and further validate their influence in driving metastasis. This study received funding from DOD grant BC151687. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). IM number: LLNL-POST-758941 Citation Format: Kelly A. Martin, Nicholas R. Hum, Aimy Sebastian, Gabriela G. Loots. Comparison of exosomes shed by breast cancer cell lines with varying metastatic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5157.

Preclinical cancer models have been vital contributors in minimizing this burden as well as understanding basic cancer cell biology, however conventional and modern cancer models do not accurately or reliably recapitulate the complex in vivo tumor environment. Clinical significance of discoveries made using in vitromodels requires an understanding of the limitations imparted from cancer cells in a non-native environment. An ideal pre-clinical cancer platform that mimicks in vivo molecular phenotypes is essential for achieving effective drug screening and personalized treatments. This study aims to elucidate biological processes deficient in conventional in vitro methods from in vivo grown allograft cancer cells via transcriptome analysis. The effects of culturing conditions on cancer cells were analyzed via whole transcriptome RNA sequencing on a mouse mammary carcinoma (4T1) cell line grown in multiple culture conditions: 2D (monolayer) or 3D (spheroid) constructs under static or dynamic flow in addition to 4T1 cells isolated from subcutaneous or orthotopically grown tumors into the mammary fat pad of immune-competent, BALB/c mice. Comparative analysis of whole transcriptomic profiles of 4T1 cells in differing culturing conditions reveals distinct biological processes fostered by their environment. Monolayer culture shows enrichment in gene ontologies promoting proliferation, cell cycle progression, and protein synthesis. Compared to monolayer culture all 3-dimensional culturing methods encouraged the expression of proteins known to be critical to tumor progression such as extracellular matrix remodeling, adhesion, and differentiation. Furthermore, spheroid culture introduced heterogeneity as evidenced by upregulation of hypoxic induced genes and regulation of multicellular organism development processes. As expected, 4T1 cells expanded in vivo upregulated genes associated with processes difficult to recapitulate in vitro such as cell migration, inflammatory response, and angiogenesis. 3D culturing methods are able to recapitulate aspects of tumor heterogeneity yet fail to incorporate the complex heterogeneous cell composition and transient fluxes in nutrients and drugs found in vivo. Findings from this study demonstrate the behavioral and transcriptional alterations imparted from environmental factors. Additionally, clinically relevant in vitro testing can be improved by the incorporation of factors found in the native tumor microenvironment to existing 3D culturing approaches. This study received funding from LLNL LDRD grant 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). Citation Format: Nicholas Hum, Aimy Sebastian, Wei He, Monica L. Moya, William F. Hynes, Jonathan J. Adorno, Aubree Hinckley, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. RNA-seq comparisons of in vitro and in vivo cancer model platforms: Monolayer, spheroids, immunodeficient, and syngeneic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 37.

It is currently unclear how stromal components affect drug response and the emergence of drug resistance, in primary tumors. To determine how individual cells within the stroma of triple negative breast cancer (TNBC) allografts respond to chemotherapy, we used single cell sequencing to profile individual cells present in murine tumors with or without exposure to doxorubicin (Dox). Dox is a common chemotherapeutic agent used to treat breast cancer which inhibits breast cancer proliferation by intercalating into DNA and preventing topoisomerase II activity. Several autonomous processes have been implicated in the development of chemoresistance yet the impact of stromal and immune cells on tumor progression is still poorly understood. In this study, TNBC 4T1 cell line were utilized to generate murine allograft tumors in immunocompetent BALB/c mice. Tumor composition was analyzed via single cell RNA sequencing after 3 and 7 days of doxorubicin chemotherapeutic regiment mimicking clinical treatment. Using Cell Ranger single cell software suite and Seurat R toolkit, single cell transcriptomic analysis identified the cellular composition of tumors through expression of cell-type specific genes. Stromal cell types such as endothelial, fibroblast and epithelial cells were assessed and quantified in the tumor microenvironment. Immune cell types including neutrophils, monocytes, macrophages, T-cells and B-cells were also identified in the stroma and the responses to doxorubicin treatment was determined based on the gene expression changes. In this study, cancer-associated fibroblasts and non-canonical tumor associated macrophage subpopulations are of particular interest. As expected, we found both qualitative and quantitative changes in specific subpopulations of stromal cells in response to Dox exposure. Identification of stromal and immune cell sub-types could also lead to improved diagnostic capabilities and tumor susceptibilities. Future studies modulating non-cancerous cells in the tumor microenvironment may increase efficacy of chemotherapeutics. Further elucidating the specific cellular subpopulations within the tumor microenvironment that shift in response to drug exposure may provide new therapeutic avenues. Understanding changes in cell populations within the drug exposed tumor microenvironment can aid in future drug development to specifically target cells least sensitive to chemotherapy exposure. This study received funding from LLNL LDRD grant 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). Citation Format: Nicholas Hum, Aimy Sebastian, Sean Gilmore, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. Characterization of the tumor microenvironment using single cell transcriptomics of triple negative breast cancer allografts treated with doxorubicin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 130.

Rift Valley Fever Virus (RVFV) is a category A biodefense pathogen due to the potential for intentional spread by aerosol and the lack of any licensed vaccine or antiviral therapeutics. Despite the brain being a major target of viral replication and tissue damage following aerosol infection, little is known about innate immune responses to RVFV in the brain. Thus, we are investigating the responses of resident innate immune cells of the brain to RVFV infection. Microglia and astrocytes mount a robust response to RVFV infection that is dependent upon MAVS and independent of TLR3 and TLR7. MAVS KO mice have significantly higher viral titers in the brain following intranasal infection, but surprisingly many markers of immune activity were similarly upregulated in the brains of WT and KO mice, including infiltration of CD45+ cells, upregulation of inflammatory genes, and cytokine expression. Indeed, many inflammatory and cytokine genes were upregulated more significantly in infected KO brains than in WT. This is in contrast to the in vitro response of microglia and astrocytes, where MAVS KO cells were virtually unresponsive to RVFV infection. RNA-seq analysis of infected brain tissue revealed that key signal transduction pathways are not activated in MAVS KO animals. These results are an important step towards understanding the precise molecular pathways responsible for controlling RVFV infection in the brain, and will be critical to informing the development of vaccines and antiviral therapeutics that are successful in preventing encephalitis caused by RVFV.

Sclerostin is an osteocyte derived negative regulator of bone formation. A highly specific expression pattern and the exclusive bone phenotype have made Sclerostin an attractive target for therapeutic intervention in treating metabolic bone diseases such as osteoporosis and in facilitating fracture repair. Understanding the molecular mechanisms that regulate Sclerostin transcription is of great interest as it may unveil new avenues for therapeutic approaches. Such studies may also elucidate how various signaling pathways intersect to modulate bone metabolism. Here we review the current understanding of the upstream molecular mechanisms that regulate Sost/SOST transcription, in bone.

Joint injury causes post‐traumatic osteoarthritis (PTOA). About ∼50% of patients rupturing their anterior cruciate ligament (ACL) will develop PTOA within 1–2 decades of the injury, yet the mechanisms responsible for the development of PTOA after joint injury are not well understood. In this study, we examined whole joint gene expression by RNA sequencing (RNAseq) at 1 day, 1‐, 6‐, and 12 weeks post injury, in a non‐invasive tibial compression (TC) overload mouse model of PTOA that mimics ACL rupture in humans. We identified 1446 genes differentially regulated between injured and contralateral joints. This includes known regulators of osteoarthritis such as MMP3, FN1, and COMP, and several new genes including Suco, Sorcs2, and Medag. We also identified 18 long noncoding RNAs that are differentially expressed in the injured joints. By comparing our data to gene expression data generated using the surgical destabilization of the medial meniscus (DMM) PTOA model, we identified several common genes and shared mechanisms. Our study highlights several differences between these two models and suggests that the TC model may be a more rapidly progressing model of PTOA. This study provides the first account of gene expression changes associated with PTOA development and progression in a TC model. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. J Orthop Res 35:474–485, 2017.

Inhibitors of Wnt signaling have been shown to be involved in prostate cancer (PC) metastasis; however the role of Sclerostin (Sost) has not yet been explored. Here we show that elevated Wnt signaling derived from Sost deficient osteoblasts promotes PC invasion, while rhSOST has an inhibitory effect. In contrast, rhDKK1 promotes PC elongation and filopodia formation, morphological changes characteristic of an invasive phenotype. Furthermore, rhDKK1 was found to activate canonical Wnt signaling in PC3 cells, suggesting that SOST and DKK1 have opposing roles on Wnt signaling in this context. Gene expression analysis of PC3 cells co-cultured with OBs exhibiting varying amounts of Wnt signaling identified CRIM1 as one of the transcripts upregulated under highly invasive conditions. We found CRIM1 overexpression to also promote cell-invasion. These findings suggest that bone-derived Wnt signaling may enhance PC tropism by promoting CRIM1 expression and facilitating cancer cell invasion and adhesion to bone. We concluded that SOST and DKK1 have opposing effects on PC3 cell invasion and that bone-derived Wnt signaling positively contributes to the invasive phenotypes of PC3 cells by activating CRIM1 expression and facilitating PC-OB physical interaction. As such, we investigated the effects of high concentrations of SOST in vivo. We found that PC3-cells overexpressing SOST injected via the tail vein in NSG mice did not readily metastasize, and those injected intrafemorally had significantly reduced osteolysis, suggesting that targeting the molecular bone environment may influence bone metastatic prognosis in clinical settings.

Bladder cancer is among the top ten most common cancers diagnosed, with about ~380,000 new cases and ~150,000 deaths per year reported worldwide. Platinum-based chemotherapy in combination with gemcitabine, a nucleoside analog, is a widely used treatment option for advanced bladder cancer. It has been shown that only ~50% of the patients with advanced bladder cancer respond to platinum-based therapy. Cancer cells often become non-responsive to therapy that once proved efficacious and are now drug resistant. Drug resistance represents a significant, ongoing challenge in eradicating cancer. We have employed a patient-derived bladder cancer xenograft (PDX) platform to further investigate the molecular mechanisms that contribute to gemcitabine-induced drug resistance in advanced bladder cancer. Transcriptome profiling of passage 4 bladder cancer xenograft tumors from 2 gemcitabine sensitive PDX lines (BL0440 & BL0293) was performed using RNA sequencing (RNAseq) analysis, before and after a 21-day cisplatin/gemcitabine drug treatment regimen. Key regulatory pathways and genes contributing to drug resistant bladder cancer have been identified and validated by overexpression in a 5637 bladder cancer cell line. RNA sequencing data has indicated significant differences between the transcriptional profiles of drug-sensitive and drug-resistant tumors. PDXs retained morphology and shared 92-97% of genetic alterations of parental cancer cells. We identified 106 genes >1.5 fold up-regulated and 45 genes >1.5 fold down-regulated in the drug resistant tumors compared to their drug sensitive counterparts. Of the genes that were significantly upregulated, two methyltransferase enzymes were further validated: Nicotinamide N-methyltransferase (NNMT) and methionine methyltransferase 1A (MAT1A). These genes were found to be contributors to gemcitabine-mediated drug resistance. 5637 cells overexpressing NNMT yielded a 200-fold increase in gemcitabine resistance relative to parental strain. In conclusion, chemoresistance to gemcitabine is associated with differential expression of genes and alteration of downstream signaling pathways. Upregulation of NNMT & MAT1A can potentially be used as novel biomarkers for subpopulations of drug resistant bladder cancer for which improved therapeutics can be developed. Future direction will likely include studies to elucidate exact molecular mechanisms by which cancer cells utilize methyltransferases to no longer respond to gemcitabine therapy. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). Citation Format: Kelly A. Martin, Aimy Sebastian, Nicholas R. Hum, Deepa K. Murugesh, Chong-xian Pan, Ai-Hong Ma, Ralph W. de Vere White, Gabriela G. Loots. Overexpression of nicotinamide N-methyltransferase confers gemcitabine resistance in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 908.

An ideal pre-clinical environment that recapitulates in vivo growth conditions ex vivo is an essential pre-requisite for effective drug screening. Conventional monolayer culturing of cancer cells as a pre-clinical model have repeatedly failed to recapitulate responses seen clinically. While 3D culturing methods are able to generate some level of tumor heterogeneity including hypoxic core, cell-cell interactions, gradients of drug penetration, as well as cancer stem cell differentiation, these culturing methods fail to incorporate the complex heterogenous cell composition and transient fluxes in nutrients or drugs. To investigate the effects of culturing conditions on gene expression of cancer cells, RNA sequencing was performed on a mouse mammary carcinoma (4T1) cell line grown in a variety of culture conditions: 2D (monolayer) or 3D (spheroid). Additionally, gene expression analysis was performed on tumors derived from 4T1 cells injected subcutaneously (SQ) into the murine flank or orthotopically (OT) into the mammary fat pad of BALB/c mice. Pairwise analysis of RNA sequencing data identified 235 down- and 1029 up- regulated genes differentially expressed between the 2D and 3D culture methods. Differential expression identified genes involved in cell migration, extracellular matrix organization, cell adhesion, angiogenesis, hypoxic response, cell differentiation, as well as key cancer related pathways including TNF, Jak-STAT, and PI3K-AKt primarily upregulated in 3D culture. Similar differential expression was found for genes encoding extracellular matrix proteins, which may be reflective of the 3D environment. Both in vivo allograft models produced highly similar gene expression profiles with only 31 up- and 20 down- regulated genes differentially expressed between OT and SQ tumors. Down-regulated genes were enriched in transcriptional regulatory gene networks and up-regulated genes were enriched for signaling/secreted proteins. The gene expression profiles of in vivo tumors were significantly different when compared to the 2D (973 down regulated, 1971 upregulated genes); and 99 of these transcripts were only expressed in in vivo tumors, highlighting the increased heterogeneity of cell composition found in allografts. 3D culturing of cancer cells upregulates pathways known to be critical to tumor progression including genes known to be essential for adhesion, differentiation, and ECM remodeling however the gene expression profile of spheroids is significantly different than that of cancer cells expanded in vivo. Future culturing methods incorporating immune cells, cancer supporting cells such as fibroblasts and other stromal components are more likely to improve the phenotype of 3D cultured tumor cells. This study received funding from LLNL LDRD grant 17-ERD-121. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). Citation Format: Nicholas R. Hum, Wei He, Aimy Sebastian, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. Determining gene expression variability between in vitro and in vivo cancer models: Monolayer, spheroids, and mouse allografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2528.

Human Protein Reference Database (HPRD--http://www.hprd.org/), initially described in 2003, is a database of curated proteomic information pertaining to human proteins. We have recently added a number of new features in HPRD. These include PhosphoMotif Finder, which allows users to find the presence of over 320 experimentally verified phosphorylation motifs in proteins of interest. Another new feature is a protein distributed annotation system--Human Proteinpedia (http://www.humanproteinpedia.org/)--through which laboratories can submit their data, which is mapped onto protein entries in HPRD. Over 75 laboratories involved in proteomics research have already participated in this effort by submitting data for over 15,000 human proteins. The submitted data includes mass spectrometry and protein microarray-derived data, among other data types. Finally, HPRD is also linked to a compendium of human signaling pathways developed by our group, NetPath (http://www.netpath.org/), which currently contains annotations for several cancer and immune signaling pathways. Since the last update, more than 5500 new protein sequences have been added, making HPRD a comprehensive resource for studying the human proteome.

Background: Bladder cancer is among the top ten most common cancers, with about ~380,000 new cases and ~150,000 deaths per year worldwide. Platinum-based combination chemotherapy is commonly used to treat advanced bladder cancer. It has been shown that only ~50% of the patients with advanced bladder cancer respond to platinum-based therapy. Methods: We have utilized a patient-derived bladder cancer xenograft (PDX) platform to characterize the molecular mechanisms that contribute to resistance of gemcitabine-cisplatin combination therapy in advanced bladder cancer. We have also identified key regulatory pathways in our PDX models that can be targeted to treat chemotherapy resistant bladder cancer using RNAseq analysis. Transcriptome profiling of P0 (passage 0) bladder cancer xenograft tumors from 4 PDX lines (2 gemcitabine-cisplatin resistant lines and 2 drug sensitive lines) was performed by RNA-Seq analysis, before and after a 21-day cisplatin/gemcitabine drug treatment regimen. Results: The RNA-seq data has indicated significant differences between the transcriptional profiles of drug-sensitive and drug-resistant tumors. PDXs retained morphology and shared 92-97% of genetic alterations of parental cancer cells. We identified 333 genes >2 fold up or down regulated in the drug resistant tumors compared to the drug sensitive tumors. Significantly up-regulated genes in drug resistant tumors analyzed include metabolic enzymes ALDH2, ALDH3A1, ALDH4A1 and ALDH7A1, transporter proteins ABCA1, SLC1A4, SLC2A5, SLC30A1, SLC39A6, SLC7A5 and SLC9A3, Notch ligand JAG2, Growth hormone receptor GHR and transmembrane protein GPNMB. Consistent with the change of cell surface proteins such as GHR and GPNMB, the MAPK and the PI3K-AKT pathways were upregulated when PDXs became resistant to cisplatin treatment. Additional changes in gene expression based on RNA-seq data before and after drug treatment were also found. Conclusion: Chemoresistance to gemcitabine and cisplatin is associated with altered expression of several cell surface proteins and upregulation of the downstream signaling pathways. Targeting these cell surface proteins can possibly be harnessed to overcome chemoresistance. GPNMB is a type I transmembrane protein that has previously been shown to be up-regulated in many metastatic cancers including breast cancer. Potentially targeting GPNMB with glembatumumab vedotin, an antibody-drug-conjugate has shown promising results in treating several cancers including breast cancer and osteosarcoma. Further studies will elucidate whether targeting GPNMB is an effective strategy for the treatment of chemotherapy resistant bladder cancer. This study received funding by a developmental grant from the UCDCC. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). IM number: LLNL-688318 Citation Format: Kelly A. Martin, Nicholas R. Hum, Aimy Sebastian, Deepa K. Murugesh, Chong-Xian Pan, Ai-Hong Ma, Ralph de Vere White, Gaby Loots. RNA sequencing of bladder cancer patient-derived xenograft models identifies genes associated with chemoresistance [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 97. doi:10.1158/1538-7445.AM2017-97

Background: Bladder cancer is among the ten most common cancers, with about ∼380,000 new cases and ∼150,000 deaths per year worldwide. Platinum-based combination chemotherapy is commonly used to treat advanced bladder cancer. It has been shown that only ∼50% of the patients with advanced bladder cancer respond to platinum-based therapy. Methods: We employed a patient-derived bladder cancer xenograft (PDX) platform to characterize the molecular mechanisms contributing to resistance to gemcitabine-cisplatin combination therapy in advanced bladder cancer and to identify novel candidates that can be targeted to treat chemotherapy resistant bladder cancer. Transcriptome profiling of P0 (passage 0) bladder cancer xenograft tumors from 4 PDX lines (2 gemcitabine-cisplatin resistant lines and 2 drug sensitive lines) was performed by RNA-Seq analysis. Results: PDXs retained the morphology fidelity and shared 92-97% of genetic alterations of parental cancer cells. The RNA-seq data suggested the presence of significant differences between the transcription profiles of drug-sensitive and drug-resistant tumors. We identified 333 genes >2 fold up or down regulated in the drug resistant tumors compared to the drug sensitive tumors. Genes down-regulated in drug resistant tumors include tight junction protein CLDN3 and regulators of G-protein signaling RGS2 and RGS3. Significantly up-regulated genes include metabolic enzymes ALDH2, ALDH3A1, ALDH4A1 and ALDH7A1, transporter proteins ABCA1, SLC1A4, SLC2A5, SLC30A1, SLC39A6, SLC7A5 and SLC9A3, Notch ligand JAG2, Growth hormone receptor GHR and transmembrane glycoprotein GPNMB. Consistent with the change of cell surface proteins such as GHR and GPNMB, the MAPK and the PI3K-AKT pathways were upregulated when PDXs became resistant to cisplatin treatment. Conclusion: Chemoresistance to gemcitabine and cisplatin is associated with altered expression of several cell surface proteins and upregulation of the downstream signaling pathways. Targeting these cell surface proteins can possibly be harnessed to overcome chemoresistance. GPNMB, a type I transmembrane protein highly up-regulated in the drug resistant tumors, has previously been shown to be over-expressed in various cancers. Targeting GPNMB with an antibody-drug-conjugate, glembatumumab vedotin, has shown promising results in treating several cancers including breast cancer and osteosarcoma. Further studies will elucidate whether targeting GPNMB is an effective strategy for the treatment of chemotherapy resistant bladder cancer. Citation Format: Aimy Sebastian, Kelly A. Martin, Chong-xian Pan, Ai-hong Ma, Ralph W. deVere White, Gabriela G. Loots. Transcriptome analysis of patient-derived bladder cancer xenografts identifies genes associated with chemoresistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2106.

WNT signaling is critical in most aspects of skeletal development and homeostasis, and antagonists of WNT signaling are emerging as key regulatory proteins with great promise as therapeutic agents for bone disorders. Here we show that Sost and its paralog Sostdc1 emerged through ancestral genome duplication and their expression patterns have diverged to delineate non-overlapping domains in most organ systems including musculoskeletal, cardiovascular, nervous, digestive, reproductive and respiratory. In the developing limb, Sost and Sostdc1 display dynamic expression patterns with Sost being restricted to the distal ectoderm and Sostdc1 to the proximal ectoderm and the mesenchyme. While Sostdc1(-/-) mice lack any obvious limb or skeletal defects, Sost(-/-) mice recapitulate the hand defects described for Sclerosteosis patients. However, elevated WNT signaling in Sost(-/-); Sostdc1(-/-) mice causes misregulation of SHH signaling, ectopic activation of Sox9 in the digit 1 field and preaxial polydactyly in a Gli1- and Gli3-dependent manner. In addition, we show that the syndactyly documented in Sclerosteosis is present in both Sost(-/-) and Sost(-/-); Sostdc1(-/-) mice, and is driven by misregulation of Fgf8 in the AER, a region lacking Sost and Sostdc1 expression. This study highlights the complexity of WNT signaling in skeletal biology and disease and emphasizes how redundant mechanism and non-cell autonomous effects can synergize to unveil new intricate phenotypes caused by elevated WNT signaling.