Publications by authors named "Timothy L Ratliff"

97 Publications

Folate Receptor Beta Designates Immunosuppressive Tumor-Associated Myeloid Cells That Can Be Reprogrammed with Folate-Targeted Drugs.

Cancer Res 2021 02 17;81(3):671-684. Epub 2020 Nov 17.

Department of Chemistry, Purdue University, West Lafayette, Indiana.

Although immunotherapies of tumors have demonstrated promise for altering the progression of malignancies, immunotherapies have been limited by an immunosuppressive tumor microenvironment (TME) that prevents infiltrating immune cells from performing their anticancer functions. Prominent among immunosuppressive cells are myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) that inhibit T cells via release of immunosuppressive cytokines and engagement of checkpoint receptors. Here, we explore the properties of MDSCs and TAMs from freshly isolated mouse and human tumors and find that an immunosuppressive subset of these cells can be distinguished from the nonimmunosuppressive population by its upregulation of folate receptor beta (FRβ) within the TME and its restriction to the TME. This FRβ subpopulation could be selectively targeted with folate-linked drugs. Delivery of a folate-targeted TLR7 agonist to these cells (i) reduced their immunosuppressive function, (ii) increased CD8 T-cell infiltration, (iii) enhanced M1/M2 macrophage ratios, (iv) inhibited tumor growth, (v) blocked tumor metastasis, and (vi) improved overall survival without demonstrable toxicity. These data reveal a broadly applicable strategy across tumor types for reprogramming MDSCs and TAMs into antitumorigenic immune cells using a drug that would otherwise be too toxic to administer systemically. The data also establish FRβ as the first marker that distinguishes immunosuppressive from nonimmunosuppressive subsets of MDSCs and TAMs. Because all solid tumors accumulate MDSCs and TAMs, a general strategy to both identify and reprogram these cells should be broadly applied in the characterization and treatment of multiple tumors. SIGNIFICANCE: FRβ serves as both a means to identify and target MDSCs and TAMs within the tumor, allowing for delivery of immunomodulatory compounds to tumor myeloid cells in a variety of cancers.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-1414DOI Listing
February 2021

Pharmacologic Inhibition of FGFR Modulates the Metastatic Immune Microenvironment and Promotes Response to Immune Checkpoint Blockade.

Cancer Immunol Res 2020 12 22;8(12):1542-1553. Epub 2020 Oct 22.

Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana.

The effectiveness of immunotherapy as a treatment for metastatic breast cancer is limited due to low numbers of infiltrating lymphocytes in metastatic lesions. Herein, we demonstrated that adjuvant therapy using FIIN4, a covalent inhibitor of fibroblast growth factor receptor (FGFR), dramatically delayed the growth of pulmonary metastases in syngeneic models of metastatic breast cancer. In addition, we demonstrated in a syngeneic model of systemic tumor dormancy that targeting of FGFR enhanced the immunogenicity of the pulmonary tumor microenvironment through increased infiltration of CD8 lymphocytes and reduced presence of myeloid suppressor cells. Similar impacts on immune cell infiltration were observed upon genetic depletion of FGFR1 in tumor cells, which suggested a direct influence of FGFR signaling on lymphocyte trafficking. Suppression of CD8 lymphocyte infiltration was consistent with FGFR-mediated inhibition of the T-cell chemoattractant CXCL16. Initial attempts to concomitantly administer FIIN4 with immune checkpoint blockade failed due to inhibition of immune-mediated tumor cell killing via blockade of T-cell receptor signaling by FIIN4. However, this was overcome by using a sequential dosing protocol that consisted of FIIN4 treatment followed by anti-PD-L1. These data illustrate the complexities of combining kinase inhibitors with immunotherapy and provide support for further assessment of FGFR targeting as an approach to enhance antitumor immunity and improve immunotherapy response rates in patients with metastatic breast cancer.
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710538PMC
December 2020

Thrombin-PAR1 signaling in pancreatic cancer promotes an immunosuppressive microenvironment.

J Thromb Haemost 2021 01 25;19(1):161-172. Epub 2020 Oct 25.

Department of Biological Sciences and The Purdue Center for Cancer Research, Purdue University, West Lafayette, IN, USA.

Essentials Elimination of PDAC tumor cell PAR1 increased cytotoxic T cells and reduced tumor macrophages. PAR1 PDAC cells are preferentially eliminated from growing tumors. Thrombin-PAR1 signaling in PDAC tumor cells drives an immunosuppressive gene signature. Csf2 and Ptgs2 are thrombin-PAR1 downstream immune suppressor genes in PDAC tumor cells. ABSTRACT: Background Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prothrombotic state and a lack of host antitumor immune responsiveness. Linking these two key features, we previously demonstrated that tumor-derived coagulation activity promotes immune evasion. Specifically, thrombin-protease-activated receptor-1 (PAR1) signaling in mouse PDAC cells drives tumor growth by evading cytotoxic CD8a cells. Methods Syngeneic mixed cell tumor growth, transcriptional analyses, and functional tests of immunosuppressive response genes were used to identify cellular and molecular immune evasion mechanisms mediated by thrombin-PAR-1 signaling in mouse PDAC tumor cells. Results Elimination of tumor cell PAR1 in syngeneic graft studies increased cytotoxic T lymphocyte (CTL) infiltration and decreased tumor-associated macrophages in the tumor microenvironment. Co-injection of PAR1-expressing and PAR1-knockout (PAR-1 ) tumor cells into immunocompetent mice resulted in preferential elimination of PAR-1 cells from developing tumors, suggesting that PAR1-dependent immune evasion is not reliant on CTL exclusion. Transcriptomics analyses revealed no PAR1-dependent changes in the expression of immune checkpoint proteins and no difference in major histocompatibility complex-I cell surface expression. Importantly, thrombin-PAR1 signaling in PDAC cells upregulated genes linked to immunosuppression, including Csf2 and Ptgs2. Functional analyses confirmed that both Csf2 and Ptgs2 are critical for PDAC syngeneic graft tumor growth and overexpression of each factor partially restored tumor growth of PAR1 cells in immunocompetent mice. Conclusions Our results provide novel insight into the mechanisms of a previously unrecognized pathway coupling coagulation to PDAC immune evasion by identifying PAR1-dependent changes in the tumor microenvironment, a PAR1-driven immunosuppressive gene signature, and Csf2 and Ptgs2 as critical PAR1 downstream targets.
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http://dx.doi.org/10.1111/jth.15115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790967PMC
January 2021

Protein Arginine Methyltransferase 5 Promotes pICln-Dependent Androgen Receptor Transcription in Castration-Resistant Prostate Cancer.

Cancer Res 2020 11 30;80(22):4904-4917. Epub 2020 Sep 30.

Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana.

The majority of advanced prostate cancer therapies aim to inhibit androgen receptor (AR) signaling. However, AR reactivation inevitably drives disease progression to castration-resistant prostate cancer (CRPC). Here we demonstrate that protein arginine methyltransferase 5 (PRMT5) functions as an epigenetic activator of transcription in CRPC, requiring cooperation with a methylosome subunit pICln. and in xenograft tumors in mice, targeting PRMT5 or pICln suppressed growth of CRPC cells. Full-length AR and AR-V7 transcription activation required both PRMT5 and pICln but not MEP50. This activation of transcription was accompanied by PRMT5-mediated symmetric dimethylation of H4R3 at the proximal promoter. Further, knockdown of PRMT5 abolished the binding of pICln (but not vice versa) to the proximal promoter region, suggesting that PRMT5 recruits pICln to the promoter to activate transcription. Differential gene expression analysis in 22Rv1 cells confirmed that PRMT5 and pICln both regulate the androgen signaling pathway. In addition, PRMT5 and pICln protein expression positively correlated with AR and AR-V7 protein expression in CRPC tissues and their expression was highly correlated at the mRNA level across multiple publicly available CRPC datasets. Our results suggest that targeting PRMT5 or pICln may be explored as a novel therapy for CRPC treatment by suppressing expression of AR and AR splice variants to circumvent AR reactivation. SIGNIFICANCE: This study provides evidence that targeting PRMT5 can eliminate expression of AR and can be explored as a novel therapeutic approach to treat metastatic hormone-naïve and castration-resistant prostate cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-1228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7669631PMC
November 2020

Naturally-Occurring Invasive Urothelial Carcinoma in Dogs, a Unique Model to Drive Advances in Managing Muscle Invasive Bladder Cancer in Humans.

Front Oncol 2019 21;9:1493. Epub 2020 Jan 21.

Department of Oncology and Urology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, United States.

There is a great need to improve the outlook for people facing urinary bladder cancer, especially for patients with invasive urothelial carcinoma (InvUC) which is lethal in 50% of cases. Improved outcomes for patients with InvUC could come from advances on several fronts including emerging immunotherapies, targeted therapies, and new drug combinations; selection of patients most likely to respond to a given treatment based on molecular subtypes, immune signatures, and other characteristics; and prevention, early detection, and early intervention. Progress on all of these fronts will require clinically relevant animal models for translational research. The animal model(s) should possess key features that drive success or failure of cancer drugs in humans including tumor heterogeneity, genetic-epigenetic crosstalk, immune cell responsiveness, invasive and metastatic behavior, and molecular subtypes (e.g., luminal, basal). Experimental animal models, while essential in bladder cancer research, do not possess these collective features to accurately predict outcomes in humans. These key features, however, are present in naturally-occurring InvUC in pet dogs. Canine InvUC closely mimics muscle-invasive bladder cancer in humans in cellular and molecular features, molecular subtypes, immune response patterns, biological behavior (sites and frequency of metastasis), and response to therapy. Thus, dogs can offer a highly relevant animal model to complement other models in research for new therapies for bladder cancer. Clinical treatment trials in pet dogs with InvUC are considered a win-win-win scenario; the individual dog benefits from effective treatment, the results are expected to help other dogs, and the findings are expected to translate to better treatment outcomes in humans. In addition, the high breed-associated risk for InvUC in dogs (e.g., 20-fold increased risk in Scottish Terriers) offers an unparalleled opportunity to test new strategies in primary prevention, early detection, and early intervention. This review will provide an overview of canine InvUC, summarize the similarities (and differences) between canine and human InvUC, and provide evidence for the expanding value of this canine model in bladder cancer research.
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http://dx.doi.org/10.3389/fonc.2019.01493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6985458PMC
January 2020

Heterogeneity of human prostate carcinoma-associated fibroblasts implicates a role for subpopulations in myeloid cell recruitment.

Prostate 2020 02 25;80(2):173-185. Epub 2019 Nov 25.

Department of Surgery, NorthShore University HealthSystem, Evanston, Illinois.

Background: Carcinoma-associated fibroblasts (CAF) are a heterogeneous group of cells within the tumor microenvironment (TME) that can promote tumorigenesis in the prostate. By understanding the mechanism(s) by which CAF contributes to tumor growth, new therapeutic targets for the management of this disease may be identified. These studies determined whether unique sub-populations of human prostate CAF can be identified and functionally characterized.

Methods: Single-cell RNA-seq of primary human prostate CAF followed by unsupervised clustering was utilized to generate cell clusters based on differentially expressed (DE) gene profiles. Potential communication between CAF and immune cells was analyzed using in vivo tissue recombination by combining CAF or normal prostate fibroblasts (NPF) with non-tumorigenic, initiated prostate epithelial BPH-1 cells. Resultant grafts were assessed for inflammatory cell recruitment.

Results: Clustering of 3321 CAF allows for visualization of six subpopulations, demonstrating heterogeneity within CAF. Sub-renal capsule recombination assays show that the presence of CAF significantly increases myeloid cell recruitment to resultant tumors. This is supported by significantly increased expression of chemotactic chemokines CCL2 and CXCL12 in large clusters compared to other subpopulations. Bayesian analysis topologies also support differential communication signals between chemokine-related genes of individual clusters. Migration of THP-1 monocyte cells in vitro is stimulated in the presence of CAF conditioned medium (CM) compared with NPF CM. Further in vitro analyses suggest that CAF-derived chemokine CCL2 may be responsible for CAF-stimulated migration of THP-1 cells, since neutralization of this chemokine abrogates migration capacity.

Conclusions: CAF clustering based on DE gene expression supports the concept that clusters have unique functions within the TME, including a role in immune/inflammatory cell recruitment. These data suggest that CCL2 produced by CAF may be involved in the recruitment of inflammatory cells, but may also directly regulate the growth of the tumor. Further studies aimed at characterizing the subpopulation(s) of CAF which promote immune cell recruitment to the TME and/or stimulate prostate cancer growth and progression will be pursued.
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http://dx.doi.org/10.1002/pros.23929DOI Listing
February 2020

Sustained Delivery of Carfilzomib by Tannic Acid-Based Nanocapsules Helps Develop Antitumor Immunity.

Nano Lett 2019 11 4;19(11):8333-8341. Epub 2019 Nov 4.

Department of Industrial and Physical Pharmacy , Purdue University , 575 Stadium Mall Drive , West Lafayette , Indiana 47907 , United States.

A group of chemotherapeutic drugs has gained increasing interest in cancer immunotherapy due to the potential to induce immunogenic cell death (ICD). A critical challenge in using the ICD inducers in cancer immunotherapy is the immunotoxicity accompanying their antiproliferative effects. To alleviate this, a nanocapsule formulation of carfilzomib (CFZ), an ICD-inducing proteasome inhibitor, was developed using interfacial supramolecular assembly of tannic acid (TA) and iron, supplemented with albumin coating. The albumin-coated CFZ nanocapsules (CFZ-pTA-alb) attenuated CFZ release, reducing toxicity to immune cells. Moreover, due to the adhesive nature of the TA assembly, CFZ-pTA-alb served as a reservoir of damage-associated molecular patterns released from dying tumor cells to activate dendritic cells. Upon intratumoral administration, CFZ-pTA-alb prolonged tumor retention of CFZ and showed consistently greater antitumor effects than cyclodextrin-solubilized CFZ (CFZ-CD) in B16F10 and CT26 tumor models. Unlike CFZ-CD, the locally injected CFZ-pTA-alb protected or enhanced CD8 T cell population in tumors, helped develop splenocytes with tumor-specific interferon-γ response, and delayed tumor development on the contralateral side in immunocompetent mice (but not in athymic nude mice), supporting that CFZ-pTA-alb contributed to activating antitumor immunity. This study demonstrates that sustained delivery of ICD inducers by TA-based nanocapsules is an effective way of translating local ICD induction to systemic antitumor immunity.
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http://dx.doi.org/10.1021/acs.nanolett.9b04147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6885327PMC
November 2019

A novel, safe, fast and efficient treatment for Her2-positive and negative bladder cancer utilizing an EGF-anthrax toxin chimera.

Int J Cancer 2020 01 1;146(2):449-460. Epub 2019 Nov 1.

Purdue University Center for Cancer Research, West Lafayette, IN.

Bladder cancer is the sixth most common cancer in the United States, and it exhibits an alarming 70% recurrence rate. Thus, the development of more efficient antibladder cancer approaches is a high priority. Accordingly, this work provides the basis for a transformative anticancer strategy that takes advantage of the unique characteristics of the bladder. Unlike mucin-shielded normal bladder cells, cancer cells are exposed to the bladder lumen and overexpress EGFR. Therefore, we used an EGF-conjugated anthrax toxin that after targeting EGFR was internalized and triggered apoptosis in exposed bladder cancer cells. This unique agent presented advantages over other EGF-based technologies and other toxin-derivatives. In contrast to known agents, this EGF-toxin conjugate promoted its own uptake via receptor microclustering even in the presence of Her2 and induced cell death with a LC < 1 nM. Furthermore, our data showed that exposures as short as ≈3 min were enough to commit human (T24), mouse (MB49) and canine (primary) bladder cancer cells to apoptosis. Exposure of tumor-free mice and dogs with the agent resulted in no toxicity. In addition, the EGF-toxin was able to eliminate cells from human patient tumor samples. Importantly, the administration of EGF-toxin to dogs with spontaneous bladder cancer, who had failed or were not eligible for other therapies, resulted in ~30% average tumor reduction after one treatment cycle. Because of its in vitro and in vivo high efficiency, fast action (reducing treatment time from hours to minutes) and safety, we propose that this EGF-anthrax toxin conjugate provides the basis for new, transformative approaches against bladder cancer.
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http://dx.doi.org/10.1002/ijc.32719DOI Listing
January 2020

Distinct expression patterns of SULT2B1b in human prostate epithelium.

Prostate 2019 08 18;79(11):1256-1266. Epub 2019 Jun 18.

Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana.

Background: SULT2B1b (sulfotransferase family cytosolic 2B member 1b) catalyzes the sulfate conjugation of substrates such as cholesterol and oxysterols. Our laboratory has previously shown that SULT2B1b inhibition modulates androgen receptor signaling and induces apoptosis in prostate cancer cells. However, the functions of SULT2B1b in the prostate remain poorly understood.

Methods: We characterized the expression pattern of SULT2B1b in human benign prostate hyperplasia (BPH) as well as prostate cancer to determine the relationship between SULT2B1b and prostate diseases, using immunohistochemistry, immunofluorescence staining, immunoblot, and real-time polymerase chain reaction.

Results: SULT2B1b was strongly detected in the prostate epithelium but was absent in the stroma. Significantly lower SULT2B1b was found in primary cancer cells compared with adjacent normal epithelial cells. SULT2B1b further decreased in metastatic cancer cells. Most interestingly, we found, for the first time, that SULT2B1b was much more concentrated in the luminal layer than in the basal layer in both normal prostate and BPH samples. The stronger presence of SULT2B1b in luminal epithelial cells was confirmed by costaining with luminal and basal markers and in sorted paired luminal and basal cells. SULT2B1b expression was induced with prostate organoid differentiation.

Conclusions: SULT2B1b inversely correlates with prostate cancer status, with the highest level in the normal epithelium and lowest in the advanced metastatic prostate cancer. Furthermore, SULT2B1b is mostly located within the luminal layer of the prostate epithelium, suggesting that it may be implicated in luminal differentiation.
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http://dx.doi.org/10.1002/pros.23829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064034PMC
August 2019

Cholesterol Sulfotransferase SULT2B1b Modulates Sensitivity to Death Receptor Ligand TNFα in Castration-Resistant Prostate Cancer.

Mol Cancer Res 2019 06 1;17(6):1253-1263. Epub 2019 Mar 1.

Department of Comparative Pathobiology, Purdue University, West Lafayette, Indiana.

Cholesterol sulfotransferase, SULT2B1b, has been demonstrated to modulate both androgen receptor activity and cell growth properties. However, the mechanism(s) by which SULT2B1b alters these properties within prostate cancer cells has not been described. Furthermore, specific advantages of SULT2B1b expression in prostate cancer cells are not understood. In these studies, single-cell mRNA sequencing was conducted to compare the transcriptomes of SULT2B1b knockdown (KD) versus Control KD LNCaP cells. Over 2,000 differentially expressed genes were identified along with alterations in numerous canonical pathways, including the death receptor signaling pathway. The studies herein demonstrate that SULT2B1b KD increases TNFα expression in prostate cancer cells and results in NF-κB activation in a TNF-dependent manner. More importantly, SULT2B1b KD significantly enhances TNF-mediated apoptosis in both TNF-sensitive LNCaP cells and TNF-resistant C4-2 cells. Overexpression of SULT2B1b in LNCaP cells also decreases sensitivity to TNF-mediated cell death, suggesting that SULT2B1b modulates pathways dictating the TNF sensitivity capacity of prostate cancer cells. Probing human prostate cancer patient datasets further supports this work by providing evidence that SULT2B1b expression is inversely correlated with TNF-related genes, including , and . Together, these data provide evidence that SULT2B1b expression in prostate cancer cells enhances resistance to TNF and may provide a growth advantage. In addition, targeting SULT2B1b may induce an enhanced therapeutic response to TNF treatment in advanced prostate cancer. IMPLICATIONS: These data suggest that SULT2B1b expression enhances resistance to TNF and may promote prostate cancer.
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http://dx.doi.org/10.1158/1541-7786.MCR-18-1054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548593PMC
June 2019

Identification of LIMK2 as a therapeutic target in castration resistant prostate cancer.

Cancer Lett 2019 04 1;448:182-196. Epub 2019 Feb 1.

Department of Chemistry and Purdue University Center for Cancer Research, 560 Oval Drive, West Lafayette, IN, 47907, USA. Electronic address:

This study identified LIMK2 kinase as a disease-specific target in castration resistant prostate cancer (CRPC) pathogenesis, which is upregulated in response to androgen deprivation therapy, the current standard of treatment for prostate cancer. Surgical castration increases LIMK2 expression in mouse prostates due to increased hypoxia. Similarly, human clinical specimens showed highest LIMK2 levels in CRPC tissues compared to other stages, while minimal LIMK2 was observed in normal prostates. Most notably, inducible knockdown of LIMK2 fully reverses CRPC tumorigenesis in castrated mice, underscoring its potential as a clinical target for CRPC. We also identified TWIST1 as a direct substrate of LIMK2, which uncovered the molecular mechanism of LIMK2-induced malignancy. TWIST1 is strongly associated with CRPC initiation, progression and poor prognosis. LIMK2 increases TWIST1 mRNA levels upon hypoxia; and stabilizes TWIST1 by direct phosphorylation. TWIST1 also stabilizes LIMK2 by inhibiting its ubiquitylation. Phosphorylation-dead TWIST1 acts as dominant negative and fully prevents EMT and tumor formation in vivo, thereby highlighting the significance of LIMK2-TWIST1 signaling axis in CRPC. As LIMK2 null mice are viable, targeting LIMK2 should have minimal collateral toxicity, thereby improving the overall survival of CRPC patients.
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http://dx.doi.org/10.1016/j.canlet.2019.01.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079209PMC
April 2019

Dietary Protein Restriction Reprograms Tumor-Associated Macrophages and Enhances Immunotherapy.

Clin Cancer Res 2018 12 6;24(24):6383-6395. Epub 2018 Sep 6.

Genitourinary Malignancies Program, Simon Cancer Center, Indiana University, Indianapolis, Indiana.

Purpose: Diet and healthy weight are established means of reducing cancer incidence and mortality. However, the impact of diet modifications on the tumor microenvironment and antitumor immunity is not well defined. Immunosuppressive tumor-associated macrophages (TAMs) are associated with poor clinical outcomes and are potentially modifiable through dietary interventions. We tested the hypothesis that dietary protein restriction modifies macrophage function toward antitumor phenotypes.

Experimental Design: Macrophage functional status under different tissue culture conditions and was assessed by Western blot, immunofluorescence, qRT-PCR, and cytokine array analyses. Tumor growth in the context of protein or amino acid (AA) restriction and immunotherapy, namely, a survivin peptide-based vaccine or a PD-1 inhibitor, was examined in animal models of prostate (RP-B6Myc) and renal (RENCA) cell carcinoma. All tests were two-sided.

Results: Protein or AA-restricted macrophages exhibited enhanced tumoricidal, proinflammatory phenotypes, and in two syngeneic tumor models, protein or AA-restricted diets elicited reduced TAM infiltration, tumor growth, and increased response to immunotherapies. Further, we identified a distinct molecular mechanism by which AA-restriction reprograms macrophage function via a ROS/mTOR-centric cascade.

Conclusions: Dietary protein restriction alters TAM activity and enhances the tumoricidal capacity of this critical innate immune cell type, providing the rationale for clinical testing of this supportive tool in patients receiving cancer immunotherapies.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-0980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6455918PMC
December 2018

Naturally-Occurring Canine Invasive Urothelial Carcinoma: A Model for Emerging Therapies.

Bladder Cancer 2018 Apr 26;4(2):149-159. Epub 2018 Apr 26.

Department of Veterinary Clinical Sciences, Purdue University, West Lafayette, IN, USA.

The development of targeted therapies and the resurgence of immunotherapy offer enormous potential to dramatically improve the outlook for patients with invasive urothelial carcinoma (InvUC). Optimization of these therapies, however, is crucial as only a minority of patients achieve dramatic remission, and toxicities are common. With the complexities of the therapies, and the growing list of possible drug combinations to test, highly relevant animal models are needed to assess and select the most promising approaches to carry forward into human trials. The animal model(s) should possess key features that dictate success or failure of cancer drugs in humans including tumor heterogeneity, genetic-epigenetic crosstalk, immune cell responsiveness, invasive and metastatic behavior, and molecular subtypes (e.g., luminal, basal). While it may not be possible to create these collective features in experimental models, these features are present in naturally-occurring InvUC in pet dogs. Naturally occurring canine InvUC closely mimics muscle-invasive bladder cancer in humans in regards to cellular and molecular features, molecular subtypes, biological behavior (sites and frequency of metastasis), and response to therapy. Clinical treatment trials in pet dogs with InvUC are considered a win-win scenario; the individual dog benefits from effective treatment, the results are expected to help other dogs, and the findings are expected to translate to better treatment outcomes in humans. This review will provide an overview of canine InvUC, the similarities to the human condition, and the potential for dogs with InvUC to serve as a model to predict the outcomes of targeted therapy and immunotherapy in humans.
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http://dx.doi.org/10.3233/BLC-170145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5929349PMC
April 2018

Cholesterol Esterification Inhibition Suppresses Prostate Cancer Metastasis by Impairing the Wnt/β-catenin Pathway.

Mol Cancer Res 2018 06 15;16(6):974-985. Epub 2018 Mar 15.

Interdisciplinary Life Science Program, Purdue University, West Lafayette, Indiana.

Dysregulation of cholesterol is a common characteristic of human cancers including prostate cancer. This study observed an aberrant accumulation of cholesteryl ester in metastatic lesions using Raman spectroscopic analysis of lipid droplets in human prostate cancer patient tissues. Inhibition of cholesterol esterification in prostate cancer cells significantly suppresses the development and growth of metastatic cancer lesions in both orthotopic and intracardiac injection mouse models. Gene expression profiling reveals that cholesteryl ester depletion suppresses the metastatic potential through upregulation of multiple regulators that negatively impact metastasis. In addition, Wnt/β-catenin, a vital pathway for metastasis, is downregulated upon cholesteryl ester depletion. Mechanistically, inhibition of cholesterol esterification significantly blocks secretion of Wnt3a through reduction of monounsaturated fatty acid levels, which limits Wnt3a acylation. These results collectively validate cholesterol esterification as a novel metabolic target for treating metastatic prostate cancer. .
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http://dx.doi.org/10.1158/1541-7786.MCR-17-0665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5984676PMC
June 2018

Naturally Occurring Canine Invasive Urinary Bladder Cancer: A Complementary Animal Model to Improve the Success Rate in Human Clinical Trials of New Cancer Drugs.

Int J Genomics 2017 9;2017:6589529. Epub 2017 Apr 9.

Department of Veterinary Clinical Sciences, Purdue University, West Lafayette, IN 47907, USA.

Genomic analyses are defining numerous new targets for cancer therapy. Therapies aimed at specific genetic and epigenetic targets in cancer cells as well as expanded development of immunotherapies are placing increased demands on animal models. Traditional experimental models do not possess the collective features (cancer heterogeneity, molecular complexity, invasion, metastasis, and immune cell response) critical to predict success or failure of emerging therapies in humans. There is growing evidence, however, that dogs with specific forms of naturally occurring cancer can serve as highly relevant animal models to complement traditional models. Invasive urinary bladder cancer (invasive urothelial carcinoma (InvUC)) in dogs, for example, closely mimics the cancer in humans in pathology, molecular features, biological behavior including sites and frequency of distant metastasis, and response to chemotherapy. Genomic analyses are defining further intriguing similarities between InvUC in dogs and that in humans. Multiple canine clinical trials have been completed, and others are in progress with the aim of translating important findings into humans to increase the success rate of human trials, as well as helping pet dogs. Examples of successful targeted therapy studies and the challenges to be met to fully utilize naturally occurring dog models of cancer will be reviewed.
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http://dx.doi.org/10.1155/2017/6589529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5401760PMC
April 2017

Targeting and Internalization of Liposomes by Bladder Tumor Cells Using a Fibronectin Attachment Protein-Derived Peptide-Lipopolymer Conjugate.

Bioconjug Chem 2017 05 5;28(5):1481-1490. Epub 2017 May 5.

Purdue University Center for Cancer Research, Multi-disciplinary Cancer Research Facility, Purdue University , 1203 West State Street, West Lafayette, Indiana 47907, United States.

A synthetic peptidolipopolymer conjugate, incorporated into liposomes to promote specific binding to the fibronectin (FBN) matrix surrounding bladder tumor cells and promote cellular internalization of FBN-integrin complexes, is reported. The peptide promotes association with MB49 mouse model bladder tumor cells in a sequence-specific and concentration-dependent manner, with the maximum cell association occurring at 2 mol % RWFV-PEG2000-DSPE. Double PEGylation of the liposome membrane (i.e., 4 mol % mPEG1000-DSPE + 2 mol % RWFV-PEG2000-DSPE) enhanced binding by >1.6-fold, by improving ligand presentation on the liposome surface. The sequence specificity of the peptide-lipopolymer construct was confirmed by comparing liposomes containing RWFV-PEG2000-DSPE with scrambled and nonpeptidic lipopolymer liposomal formulations. MB49 tumor-bearing mice showed greater mean radiance values for FAP peptide-targeted liposomes in tumor-associated regions of interest than for nontargeted and scrambled peptide liposome formulations. These findings suggest that peptide-modified liposomes may be an attractive vehicle for targeted delivery to bladder tumors in vivo.
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http://dx.doi.org/10.1021/acs.bioconjchem.7b00153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6502482PMC
May 2017

Pten is necessary for the quiescence and maintenance of adult muscle stem cells.

Nat Commun 2017 01 17;8:14328. Epub 2017 Jan 17.

Department of Animal Sciences, Purdue University, 901 W State Street, West Lafayette, Indiana 47907, USA.

Satellite cells (SCs) are myogenic stem cells required for regeneration of adult skeletal muscles. A proper balance among quiescence, activation and differentiation is essential for long-term maintenance of SCs and their regenerative function. Here we show a function of Pten (phosphatase and tensin homologue) in quiescent SCs. Deletion of Pten in quiescent SCs leads to their spontaneous activation and premature differentiation without proliferation, resulting in depletion of SC pool and regenerative failure. However, prior to depletion, Pten-null activated SCs can transiently proliferate upon injury and regenerate injured muscles, but continually decline during regeneration, suggesting an inability to return to quiescence. Mechanistically, Pten deletion increases Akt phosphorylation, which induces cytoplasmic translocation of FoxO1 and suppression of Notch signalling. Accordingly, constitutive activation of Notch1 prevents SC depletion despite Pten deletion. Our findings delineate a critical function of Pten in maintaining SC quiescence and reveal an interaction between Pten and Notch signalling.
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http://dx.doi.org/10.1038/ncomms14328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5247606PMC
January 2017

Targeting Plk1 to Enhance Efficacy of Olaparib in Castration-Resistant Prostate Cancer.

Mol Cancer Ther 2017 03 9;16(3):469-479. Epub 2017 Jan 9.

Department of Biochemistry, Purdue University, West Lafayette, Indiana.

Olaparib is an FDA-approved PARP inhibitor (PARPi) that has shown promise as a synthetic lethal treatment approach for BRCA-mutant castration-resistant prostate cancer (CRPC) in clinical use. However, emerging data have also shown that even BRCA-mutant cells may be resistant to PARPi. The mechanistic basis for these drug resistances is poorly understood. Polo-like kinase 1 (Plk1), a critical regulator of many cell-cycle events, is significantly elevated upon castration of mice carrying xenograft prostate tumors. Herein, by combination with Plk1 inhibitor BI2536, we show a robust sensitization of olaparib in 22RV1, a BRCA1-deficient CRPC cell line, as well as in CRPC xenograft tumors. Mechanistically, monotherapy with olaparib results in an override of the G-S checkpoint, leading to high expression of Plk1, which attenuates olaparib's overall efficacy. In BRCA1 wild-type C4-2 cells, Plk1 inhibition also significantly increases the efficacy of olaparib in the presence of p53 inhibitor. Collectively, our findings not only implicate the critical role of Plk1 in PARPi resistance in BRCA-mutant CRPC cells, but also shed new light on the treatment of non-BRCA-mutant patient subgroups who might also respond favorably to PARPi. .
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http://dx.doi.org/10.1158/1535-7163.MCT-16-0361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5337144PMC
March 2017

Separation and dual detection of prostate cancer cells and protein biomarkers using a microchip device.

Lab Chip 2017 01;17(3):415-428

School of Mechanical Engineering, Purdue University, West Lafayette, IN 47907, USA. and Birck Nanotechnology Center, Purdue University, West Lafayette, IN 47907, USA and Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA.

Current efforts for the detection of prostate cancer using only prostate specific antigen are not ideal and indicate a need to develop new assays - using multiple targets - that can more accurately stratify disease states. We previously introduced a device capable of the concurrent detection of cellular and molecular markers from a single sample fluid. Here, an improved design, which achieves affinity as well as size-based separation of captured targets using antibody-conjugated magnetic beads and a silicon chip containing micro-apertures, is presented. Upon injection of the sample, the integration of magnetic attraction with the micro-aperture chip permits larger cell-bead complexes to be isolated in an upper chamber with the smaller protein-bead complexes and remaining beads passing through the micro-apertures into the lower chamber. This enhances captured cell purity for on chip quantification, allows the separate retrieval of captured cells and proteins for downstream analysis, and enables higher bead concentrations for improved multiplexed ligand targeting. Using LNCaP cells and prostate specific membrane antigen (PSMA) to model prostate cancer, the device was able to detect 34 pM of spiked PSMA and achieve a cell capture efficiency of 93% from culture media. LNCaP cells and PSMA were then spiked into diluted healthy human blood to mimic a cancer patient. The device enabled the detection of spiked PSMA (relative to endogenous PSMA) while recovering 85-90% of LNCaP cells which illustrated the potential of new assays for the diagnosis of prostate cancer.
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http://dx.doi.org/10.1039/c6lc01279eDOI Listing
January 2017

Comparison of Digital Rectal Examination and Serum Prostate Specific Antigen in the Early Detection of Prostate Cancer: Results of a Multicenter Clinical Trial of 6,630 Men.

J Urol 2017 02 22;197(2S):S200-S207. Epub 2016 Dec 22.

Division of Urologic Surgery, Washington University School of Medicine, St. Louis, Missouri; Division of Urologic Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts; Divisions of Urology and Hematology-Oncology, University of Arizona College of Medicine and Tucson Veterans Affairs Medical Center, Tucson, Arizona; Scott Department of Urology, Baylor College of Medicine, Houston, Texas; Department of Urology, Loyola University Medical Center, Chicago, Illinois; Division of Urology, UCLA School of Medicine, Los Angeles and Department of Clinical Research, Hybritech Incorporated, San Diego, California.

To compare the efficacy of digital rectal examination and serum prostate specific antigen (PSA) in the early detection of prostate cancer, we conducted a prospective clinical trial at 6 university centers of 6,630 male volunteers 50 years old or older who underwent PSA determination (Hybritech Tandom-E or Tandem-R assays) and digital rectal examination. Quadrant biopsies were performed if the PSA level was greater than 4 μg./l. or digital rectal examination was suspicious, even if transrectal ultrasonography revealed no areas suspicious for cancer. The results showed that 15% of the men had a PSA level of greater than 4 μg./l., 15% had a suspicious digital rectal examination and 26% had suspicious findings on either or both tests. Of 1,167 biopsies performed cancer was detected in 264. PSA detected significantly more tumors (82%, 216 of 264 cancers) than digital rectal examination (55%, 146 of 264, p = 0.001). The cancer detection rate was 3.2% for digital rectal examination, 4.6% for PSA and 5.8% for the 2 methods combined. Positive predictive value was 32% for PSA and 21% for digital rectal examination. Of 160 patients who underwent radical prostatectomy and pathological staging 114 (71%) had organ confined cancer: PSA detected 85 (75%) and digital rectal examination detected 64 (56%, p = 0.003). Use of the 2 methods in combination increased detection of organ confined disease by 78% (50 of 64 cases) over digital rectal examination alone. If the performance of a biopsy would have required suspicious transrectal ultrasonography findings, nearly 40% of the tumors would have been missed. We conclude that the use of PSA in conjunction with digital rectal examination enhances early prostate cancer detection. Prostatic biopsy should be considered if either the PSA level is greater than 4 μg./l. or digital rectal examination is suspicious for cancer, even in the absence of abnormal transrectal ultrasonography findings.
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http://dx.doi.org/10.1016/j.juro.2016.10.073DOI Listing
February 2017

Novel Use of Folate-Targeted Intraoperative Fluorescence, OTL38, in Robot-Assisted Laparoscopic Partial Nephrectomy: Report of the First Three Cases.

J Endourol Case Rep 2016 1;2(1):189-197. Epub 2016 Nov 1.

Department of Urology, Indiana University School of Medicine , Indianapolis, Indiana.

Partial nephrectomy is now the preferred surgical option for small renal tumors because it allows nephron preservation without compromising oncologic clearance. Its outcomes depend on the surgeon's ability to continuously identify the edges of the tumor during resection, thus leaving an adequate margin around the tumor without excessive removal of normal parenchyma, as well as keeping a short ischemic time. Folate receptors are highly abundant in the normal kidney, and there is a difference in folate receptor expression between malignant and normal renal tissues. Thus, the use of fluorescent agents that target folate receptors should result in differential fluorescence between the tumor and surrounding parenchyma during partial nephrectomy, which, in turn, helps tumor demarcation for identification and resection. A phase 2 study on the novel use of OTL38 in robot-assisted laparoscopic partial nephrectomy is currently in progress in our institution. The outcomes of the first three cases have shown the possible advantages of OTL38 in intraoperative tumor identification before resection and recognition of residual disease in the surrounding parenchyma after resection. The tumors typically appeared dark while the surrounding parenchyma showed brighter fluorescence. Immediately after tumor resection, the margins of all the specimens appeared to have a uniformly bright fluorescence, suggestive of an intact margin of normal renal parenchyma along the plane of excision. The pattern of intraoperative fluorescence correlates well with immunohistochemistry. No OTL38-related adverse effects have been seen among these three patients. We present the outcomes of these three cases, illustrated with intraoperative and immunohistochemistry images.
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http://dx.doi.org/10.1089/cren.2016.0104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5107661PMC
November 2016

Cholesterol Sulfonation Enzyme, SULT2B1b, Modulates AR and Cell Growth Properties in Prostate Cancer.

Mol Cancer Res 2016 09 24;14(9):776-86. Epub 2016 Jun 24.

Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana. Purdue University Center for Cancer Research, West Lafayette, Indiana.

Unlabelled: Cholesterol accumulates in prostate lesions and has been linked to prostate cancer incidence and progression. However, how accumulated cholesterol contributes to prostate cancer development and progression is not completely understood. Cholesterol sulfate (CS), the primary sulfonation product of cholesterol sulfotransferase (SULT2B1b), accumulates in human prostate adenocarcinoma and precancerous prostatic intraepithelial neoplasia (PIN) lesions compared with normal regions of the same tissue sample. Given the enhanced accumulation of CS in these lesions, it was hypothesized that SULT2B1b-mediated production of CS provides a growth advantage to these cells. To address this, prostate cancer cells with RNAi-mediated knockdown (KD) of SULT2B1b were used to assess the impact on cell growth and survival. SULT2B1b is expressed and functional in a variety of prostate cells, and the data demonstrate that SULT2B1b KD, in LNCaP and other androgen-responsive (VCaP and C4-2) cells, results in decreased cell growth/viability and induces cell death. SULT2B1b KD also decreases androgen receptor (AR) activity and expression at mRNA and protein levels. While AR overexpression has no impact on SULT2B1b KD-mediated cell death, the addition of exogenous androgen is able to partially rescue the growth inhibition induced by SULT2B1b KD in LNCaP cells. These results suggest that SULT2B1b positively regulates the AR either through alterations in ligand availability or by interaction with critical coregulators that influence AR activity.

Implications: These findings provide evidence that SULT2B1b is a novel regulator of AR activity and cell growth in prostate cancer and should be further investigated for therapeutic potential. Mol Cancer Res; 14(9); 776-86. ©2016 AACR.
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http://dx.doi.org/10.1158/1541-7786.MCR-16-0137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5111871PMC
September 2016

Expression of Cationic Amino Acid Transporter 2 Is Required for Myeloid-Derived Suppressor Cell-Mediated Control of T Cell Immunity.

J Immunol 2015 Dec 21;195(11):5237-50. Epub 2015 Oct 21.

Comparative Pathobiology Department, Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907; and

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature cells that expand during benign and cancer-associated inflammation and are characterized by their ability to inhibit T cell immunity. Increased metabolism of l-Arginine (l-Arg), through the enzymes arginase 1 and NO synthase 2 (NOS2), is well documented as a major MDSC suppressive mechanism. Therefore, we hypothesized that restricting MDSC uptake of l-Arg is a critical control point to modulate their suppressor activity. Using murine models of prostate-specific inflammation and cancer, we have identified the mechanisms by which extracellular l-Arg is transported into MDSCs. We have shown that MDSCs recruited to localized inflammation and tumor sites upregulate cationic amino acid transporter 2 (Cat2), coordinately with Arg1 and Nos2. Cat2 expression is not induced in MDSCs in peripheral organs. CAT2 contributes to the transport of l-Arg in MDSCs and is an important regulator of MDSC suppressive function. MDSCs that lack CAT2 have significantly reduced suppressive ability ex vivo and display impaired capacity for regulating T cell responses in vivo as evidenced by increased T cell expansion and decreased tumor growth in Cat2(-/-) mice. The abrogation of suppressive function is due to low intracellular l-Arg levels, which leads to the impaired ability of NOS2 to catalyze l-Arg-dependent metabolic processes. Together, these findings demonstrate that CAT2 modulates MDSC function. In the absence of CAT2, MDSCs display diminished capacity for controlling T cell immunity in prostate inflammation and cancer models, where the loss of CAT2 results in enhanced antitumor activity.
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http://dx.doi.org/10.4049/jimmunol.1500959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655170PMC
December 2015

Cotargeting Polo-Like Kinase 1 and the Wnt/β-Catenin Signaling Pathway in Castration-Resistant Prostate Cancer.

Mol Cell Biol 2015 Dec 5;35(24):4185-98. Epub 2015 Oct 5.

Department of Biochemistry, Purdue University, West Lafayette, Indiana, USA Center for Cancer Research, Purdue University, West Lafayette, Indiana, USA

The Wnt/β-catenin signaling pathway has been identified as one of the predominantly upregulated pathways in castration-resistant prostate cancer (CRPC). However, whether targeting the β-catenin pathway will prove effective as a CRPC treatment remains unknown. Polo-like kinase 1 (Plk1) is a critical regulator in many cell cycle events, and its level is significantly elevated upon castration of mice carrying xenograft prostate tumors. Indeed, inhibition of Plk1 has been shown to inhibit tumor growth in several in vivo studies. Here, we show that Plk1 is a negative regulator of Wnt/β-catenin signaling. Plk1 inhibition or depletion enhances the level of cytosolic and nuclear β-catenin in human prostate cancer cells. Furthermore, inhibition of Wnt/β-catenin signaling significantly potentiates the antineoplastic activity of the Plk1 inhibitor BI2536 in both cultured prostate cancer cells and CRPC xenograft tumors. Mechanistically, axin2, a negative regulator of the β-catenin pathway, serves as a substrate of Plk1, and Plk1 phosphorylation of axin2 facilitates the degradation of β-catenin by enhancing binding between glycogen synthase kinase 3β (GSK3β) and β-catenin. Plk1-phosphorylated axin2 also exhibits resistance to Cdc20-mediated degradation. Overall, this study identifies a novel Plk1-Wnt signaling axis in prostate cancer, offering a promising new therapeutic option to treat CRPC.
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http://dx.doi.org/10.1128/MCB.00825-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4648817PMC
December 2015

Characterization of autoimmune inflammation induced prostate stem cell expansion.

Prostate 2015 Oct 14;75(14):1620-31. Epub 2015 Jul 14.

Department of Comparative Pathobiology, Purdue University, West Lafayette, Indiana.

Background: The presence of inflammation in prostate cancer (PCa) and benign prostate hyperplasia (BPH) has been well described but the cellular mechanisms by which inflammation modulates the prostate are currently unclear. Prostate stem cells (PSC) not only maintain prostate homeostasis but also are considered to be the cell of origin of PCa and an important contributor to BPH. However, the impact of inflammation on PSC is not well understood. Therefore, we initiated studies to evaluate the effect of inflammation on PSC.

Method: Ovalbumin specific CD8(+) T cells were intravenously delivered to intact and castrated prostate ovalbumin expressing transgenic-3 (POET-3) mice to induce inflammation. Lin (CD45/CD31)(-) Sca1(+) CD49f(+) cells (LSC) and progenitor cells within LSC were determined by flow cytometry. Sorted LSC were subjected to a prostate sphere forming assay to evaluate PSC clonal propagation, proliferation, immediate differentiation, and self-renewal ability. Density of individual spheres was measured by a cantilever-based resonator weighing system. Morphology and characterization of prostate spheres was determined by hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). Finally, immediate PSC differentiation in sphere formation was determined by immunofluorescence for epithelial cytokeratin markers cytokeratin (CK) 5 and CK8.

Result: Data presented here demonstrate a significant expansion of the proliferative (BrdU(+) ) LSC population, including CK5(+) , p63(+) , CK18(+) cells, as well as intermediate cells (CK5(+) /CK8(+) ) in inflamed prostates. Histological images reveal that PSC from inflamed prostates produce significantly larger spheres, indicating that the enhanced proliferation observed in LSC is sustained in vitro in the absence of inflammatory mediators. In addition, cultures from inflamed PSC yielded increased number of tubule-like spheres. These tube-like spheres grown from PSCs isolated from inflamed mice exhibited stratification of a CK8(+) luminal-like layer and a CK5(+) basal-like layer. Notably, the numbers of spheres formed by inflamed and non-inflamed PSC were equal, suggesting that even though proliferation is enhanced by inflammation, the homeostatic level of PSC is maintained.

Conclusion: Induction of inflammation promotes PSC expansion and immediate differentiation through highly proliferative progenitor cells while the homeostasis of PSC is maintained.
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http://dx.doi.org/10.1002/pros.23043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4720918PMC
October 2015

Expansion of prostate epithelial progenitor cells after inflammation of the mouse prostate.

Am J Physiol Renal Physiol 2015 Jun 29;308(12):F1421-30. Epub 2015 Apr 29.

Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana; Melvin and Bren Simon Cancer Center-Indiana Basic Urological Research Working Group, Indiana University, Indianapolis, Indiana

Prostatic inflammation is a nearly ubiquitous pathological feature observed in specimens from benign prostate hyperplasia and prostate cancer patients. The microenvironment of the inflamed prostate is highly reactive, and epithelial hyperplasia is a hallmark feature of inflamed prostates. How inflammation orchestrates epithelial proliferation as part of its repair and recovery action is not well understood. Here, we report that a novel epithelial progenitor cell population is induced to expand during inflammation. We used sphere culture assays, immunofluorescence, and flow cytometry to show that this population is increased in bacterially induced inflamed mouse prostates relative to naïve control prostates. We confirmed from previous reports that this population exclusively possesses the ability to regrow entire prostatic structures from single cell culture using renal grafts. In addition, putative progenitor cells harvested from inflamed animals have greater aggregation capacity than those isolated from naïve control prostates. Expansion of this critical cell population requires IL-1 signaling, as IL-1 receptor 1-null mice exhibit inflammation similar to wild-type inflamed animals but exhibit significantly reduced progenitor cell proliferation and hyperplasia. These data demonstrate that inflammation promotes hyperplasia in the mouse prostatic epithelium by inducing the expansion of a selected epithelial progenitor cell population in an IL-1 receptor-dependent manner. These findings may have significant impact on our understanding of how inflammation promotes proliferative diseases such as benign prostatic hyperplasia and prostate cancer, both of which depend on expansion of cells that exhibit a progenitor-like nature.
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http://dx.doi.org/10.1152/ajprenal.00488.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4469888PMC
June 2015

Avasimibe encapsulated in human serum albumin blocks cholesterol esterification for selective cancer treatment.

ACS Nano 2015 Mar 16;9(3):2420-32. Epub 2015 Feb 16.

†Weldon School of Biomedical Engineering, ‡Department of Biological Sciences, §Department of Chemistry, ⊥Center for Cancer Research, ∥Department of Comparative Pathobiology, #Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette, Indiana 47907, United States.

Undesirable side effects remain a significant challenge in cancer chemotherapy. Here we report a strategy for cancer-selective chemotherapy by blocking acyl-CoA cholesterol acyltransferase-1 (ACAT-1)-mediated cholesterol esterification. To efficiently block cholesterol esterification in cancer in vivo, we developed a systemically injectable nanoformulation of avasimibe (a potent ACAT-1 inhibitor), called avasimin. In cell lines of human prostate, pancreatic, lung, and colon cancer, avasimin significantly reduced cholesteryl ester storage in lipid droplets and elevated intracellular free cholesterol levels, which led to apoptosis and suppression of proliferation. In xenograft models of prostate cancer and colon cancer, intravenous administration of avasimin caused the concentration of avasimibe in tumors to be 4-fold higher than the IC50 value. Systemic treatment of avasimin notably suppressed tumor growth in mice and extended the length of survival time. No adverse effects of avasimin to normal cells and organs were observed. Together, this study provides an effective approach for selective cancer chemotherapy by targeting altered cholesterol metabolism of cancer cells.
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http://dx.doi.org/10.1021/nn504025aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909415PMC
March 2015

Impact of prostate inflammation on lesion development in the POET3(+)Pten(+/-) mouse model of prostate carcinogenesis.

Am J Pathol 2014 Dec 22;184(12):3176-91. Epub 2014 Nov 22.

Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana; Purdue University Center for Cancer Research, West Lafayette, Indiana. Electronic address:

Evidence linking prostatitis and prostate cancer development is contradictory. To study this link, the POET3 mouse, an inducible model of prostatitis, was crossed with a Pten-loss model of prostate cancer (Pten(+/-)) containing the ROSA26 luciferase allele to monitor prostate size. Prostatitis was induced, and prostate bioluminescence was tracked over 12 months, with lesion development, inflammation, and cytokine expression analyzed at 4, 8, and 12 months and compared with mice without induction of prostatitis. Acute prostatitis led to more proliferative epithelium and enhanced bioluminescence. However, 4 months after initiation of prostatitis, mice with induced inflammation had lower grade pre-neoplastic lesions. A trend existed toward greater development of carcinoma 12 months after induction of inflammation, including one of two mice with carcinoma developing perineural invasion. Two of 18 mice at the later time points developed lesions with similarities to proliferative inflammatory atrophy, including one mouse with associated carcinoma. Pten(+/-) mice developed spontaneous inflammation, and prostatitis was similar among groups of mice at 8 and 12 months. Analyzed as one cohort, lesion number and grade were positively correlated with prostatitis. Specifically, amounts of CD11b(+)Gr1(+) cells were correlated with lesion development. These results support the hypothesis that myeloid-based inflammation is associated with lesion development in the murine prostate, and previous bouts of CD8-driven prostatitis may promote invasion in the Pten(+/-) model of cancer.
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http://dx.doi.org/10.1016/j.ajpath.2014.08.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4258501PMC
December 2014

Cholesteryl ester accumulation induced by PTEN loss and PI3K/AKT activation underlies human prostate cancer aggressiveness.

Cell Metab 2014 Mar;19(3):393-406

Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA; Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA. Electronic address:

Altered lipid metabolism is increasingly recognized as a signature of cancer cells. Enabled by label-free Raman spectromicroscopy, we performed quantitative analysis of lipogenesis at single-cell level in human patient cancerous tissues. Our imaging data revealed an unexpected, aberrant accumulation of esterified cholesterol in lipid droplets of high-grade prostate cancer and metastases. Biochemical study showed that such cholesteryl ester accumulation was a consequence of loss of tumor suppressor PTEN and subsequent activation of PI3K/AKT pathway in prostate cancer cells. Furthermore, we found that such accumulation arose from significantly enhanced uptake of exogenous lipoproteins and required cholesterol esterification. Depletion of cholesteryl ester storage significantly reduced cancer proliferation, impaired cancer invasion capability, and suppressed tumor growth in mouse xenograft models with negligible toxicity. These findings open opportunities for diagnosing and treating prostate cancer by targeting the altered cholesterol metabolism.
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http://dx.doi.org/10.1016/j.cmet.2014.01.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3969850PMC
March 2014

Use of graphene as protection film in biological environments.

Sci Rep 2014 Feb 14;4:4097. Epub 2014 Feb 14.

1] Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States [2] Department of Physics, Purdue University, West Lafayette, Indiana 47907, United States.

Corrosion of metal in biomedical devices could cause serious health problems to patients. Currently ceramics coating materials used in metal implants can reduce corrosion to some extent with limitations. Here we proposed graphene as a biocompatible protective film for metal potentially for biomedical application. We confirmed graphene effectively inhibits Cu surface from corrosion in different biological aqueous environments. Results from cell viability tests suggested that graphene greatly eliminates the toxicity of Cu by inhibiting corrosion and reducing the concentration of Cu(2+) ions produced. We demonstrated that additional thiol derivatives assembled on graphene coated Cu surface can prominently enhance durability of sole graphene protection limited by the defects in graphene film. We also demonstrated that graphene coating reduced the immune response to metal in a clinical setting for the first time through the lymphocyte transformation test. Finally, an animal experiment showed the effective protection of graphene to Cu under in vivo condition. Our results open up the potential for using graphene coating to protect metal surface in biomedical application.
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http://dx.doi.org/10.1038/srep04097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3924215PMC
February 2014