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MicroRNAs (miRs) play an important role in the pathophysiology of endometriosis; however, the role of miR-210 in endometriosis remains unclear. This study explores the role of miR-210 and its targets, IGFBP3 and COL8A1, in ectopic lesion growth and development. Matched eutopic (EuE) and ectopic (EcE) endometrial samples were obtained for analysis from baboons and women with endometriosis. Immortalized human ectopic endometriotic epithelial cells (12Z cells) were utilized for functional assays. Endometriosis was experimentally induced in female baboons (n = 5). Human matched endometrial and endometriotic tissues were obtained from women (n = 9, 18–45 years old) with regular menstrual cycles. Quantitative reverse transcript polymerase chain reaction (RT-qPCR) analysis was performed for in vivo characterization of miR-210, IGFBP3, and COL8A1. In situ hybridization and immunohistochemical analysis were performed for cell-specific localization. Immortalized endometriotic epithelial cell lines (12Z) were utilized for in vitro functional assays. MiR-210 expression was decreased in EcE, while IGFBP3 and COL8A1 expression was increased in EcE. MiR-210 was expressed in the glandular epithelium of EuE but attenuated in those of EcE. IGFBP3 and COL8A1 were expressed in the glandular epithelium of EuE and were increased compared to EcE. MiR-210 overexpression in 12Z cells suppressed IGFBP3 expression and attenuated cell proliferation and migration. MiR-210 repression and subsequent unopposed IGFBP3 expression may contribute to endometriotic lesion development by increasing cell proliferation and migration.

Table S2. Statistical tests

Supplementary Figures and Table S1

UBE1L is the E1-like ubiquitin-activating enzyme for the IFN-stimulated gene, 15-kDa protein (ISG15). The UBE1L-ISG15 pathway was proposed previously to target lung carcinogenesis by inhibiting cyclin D1 expression. This study extends prior work by reporting that UBE1L promotes a complex between ISG15 and cyclin D1 and inhibited cyclin D1 but not other G1 cyclins. Transfection of the UBE1L-ISG15 deconjugase, ubiquitin-specific protein 18 (UBP43), antagonized UBE1L-dependent inhibition of cyclin D1 and ISG15-cyclin D1 conjugation. A lysine-less cyclin D1 species was resistant to these effects. UBE1L transfection reduced cyclin D1 protein but not mRNA expression. Cycloheximide treatment augmented this cyclin D1 protein instability. UBE1L knockdown increased cyclin D1 protein. UBE1L was independently retrovirally transduced into human bronchial epithelial and lung cancer cells. This reduced cyclin D1 expression and clonal cell growth. Treatment with the retinoid X receptor agonist bexarotene induced UBE1L and reduced cyclin D1 immunoblot expression. A proof-of-principle bexarotene clinical trial was independently examined for UBE1L, ISG15, cyclin D1, and Ki-67 immunohistochemical expression profiles in pretreatment versus post-treatment tumor biopsies. Increased UBE1L with reduced cyclin D1 and Ki-67 expression occurred in human lung cancer when a therapeutic bexarotene intratumoral level was achieved. Thus, a mechanism for UBE1L-mediated growth suppression was found by UBE1L-ISG15 preferentially inhibiting cyclin D1. Molecular therapeutic implications are discussed. [Mol Cancer Ther 2008;7(12):3780–8]

Purpose: High-throughput profiling experiments have linked altered expression of microRNAs (miRNA) to different types of cancer. Tumor tissues are a heterogeneous mixture of not only cancer cells, but also supportive and reactive tumor microenvironment elements. To clarify the clinical significance of altered miRNA expression in solid tumors, we developed a sensitive fluorescence-based in situ hybridization (ISH) method to visualize miRNA accumulation within individual cells in formalin-fixed, paraffin-embedded tissue specimens. This ISH method was implemented to be compatible with routine clinical immunohistochemical (IHC) assays to enable the detection of miRNAs and protein markers in the same tissue section for colocalization and functional studies.Experimental Design: We used this combined ISH/IHC assay to study a subset of cancer-associated miRNAs, including miRNAs frequently detected at low (miR-34a and miR-126) and high (miR-21 and miR-155) levels, in a panel of breast, colorectal, lung, pancreas, and prostate carcinomas.Results: Despite the distinct histopathologic alterations of each particular cancer type, general trends emerged that pinpointed distinct source cells of altered miRNA expression. Although altered expressions of miR-21 and miR-34a were manifested within cancer cells, those of miR-126 and miR-155 were predominantly confined to endothelial cells and immune cells, respectively. These results suggest a heterogeneous participation of miRNAs in carcinogenesis by intrinsically affecting cancer cell biology or by modulating stromal, vascular, and immune responses.Conclusions: We described a rapid and sensitive multicolor ISH/IHC assay and showed that it could be broadly applied as an investigational tool to better understand the etiologic relevance of altered miRNA expression in cancer. Clin Cancer Res; 16(16); 4246–55. ©2010 AACR.

Supplementary Figures S1-S7 and Supplementary Tables S1-S3.

Supplementary Data from Uncovering Growth-Suppressive MicroRNAs in Lung Cancer

CCR Translation for This Article from MicroRNA-10b Expression Correlates with Response to Neoadjuvant Therapy and Survival in Pancreatic Ductal Adenocarcinoma

Purpose: MicroRNA (miRNA) expression profiles improve classification, diagnosis, and prognostic information of malignancies, including lung cancer. This study uncovered unique growth-suppressive miRNAs in lung cancer.Experimental Design: miRNA arrays were done on normal lung tissues and adenocarcinomas from wild-type and proteasome degradation-resistant cyclin E transgenic mice to reveal repressed miRNAs in lung cancer. Real-time and semiquantitative reverse transcription-PCR as well as in situ hybridization assays validated these findings. Lung cancer cell lines were derived from each transgenic line (designated as ED-1 and ED-2 cells, respectively). Each highlighted miRNA was independently transfected into these cells. Growth-suppressive mechanisms were explored. Expression of a computationally predicted miRNA target was examined. These miRNAs were studied in a paired normal-malignant human lung tissue bank.Results: miR-34c, miR-145, and miR-142-5p were repressed in transgenic lung cancers. Findings were confirmed by real-time and semiquantitative reverse transcription-PCR as well as in situ hybridization assays. Similar miRNA profiles occurred in human normal versus malignant lung tissues. Individual overexpression of miR-34c, miR-145, and miR-142-5p in ED-1 and ED-2 cells markedly repressed cell growth. Anti-miR cotransfections antagonized this inhibition. The miR-34c target, cyclin E, was repressed by miR-34c transfection and provided a mechanism for observed growth suppression.Conclusions: miR-34c, miR-145, and miR-142-5p were repressed in murine and human lung cancers. Transfection of each miRNA significantly repressed lung cancer cell growth. Thus, these miRNAs were growth suppressive and are proposed to exert antineoplastic effects in the lung.

XLS file - 76K, Transcriptome comparison of tumor DCs sorted from ovarian cancer-bearing mice treated with CD40 agonists plus PEI-complexed Dsi (Dmi155 vs. GFP Dsi)

PDF file - 20KB, The suppressive effect of VISTA is stable. Bulk CD4 T cells were purified from human PBMCs by magnetic bead selection. Cells were CFSE labeled and stimulated for 3 days with 2.5ug/ml anti-CD3 co-coated with 0, 2.5, 5 or 10ug/ml of VISTA-Ig. Control-Ig was used to bring the concentration of fusion protein to 10ug/ml in all wells. Cells were then moved to anti-CD3 in the absence of fusion protein. Percentage CFSE low cells are shown as mean +/- SD. Data is representative of at least two separate experiments.

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Supplementary Methods and Materials, Figures 1-7 from Altered MicroRNA Expression Confined to Specific Epithelial Cell Subpopulations in Breast Cancer

V-domain Ig suppressor of T cell activation (VISTA) is a potent negative regulator of T-cell function that is expressed on hematopoietic cells. VISTA levels are heightened within the tumor microenvironment, in which its blockade can enhance antitumor immune responses in mice. In humans, blockade of the related programmed cell death 1 (PD-1) pathway has shown great potential in clinical immunotherapy trials. Here, we report the structure of human VISTA and examine its function in lymphocyte negative regulation in cancer. VISTA is expressed predominantly within the hematopoietic compartment with highest expression within the myeloid lineage. VISTA-Ig suppressed proliferation of T cells but not B cells and blunted the production of T-cell cytokines and activation markers. Our results establish VISTA as a negative checkpoint regulator that suppresses T-cell activation, induces Foxp3 expression, and is highly expressed within the tumor microenvironment. By analogy to PD-1 and PD-L1 blockade, VISTA blockade may offer an immunotherapeutic strategy for human cancer. Cancer Res; 74(7); 1924–32. ©2013 AACR.

PDF file - 90KB, Human and mouse VISTA-Ig show titratable suppressive activity on human CD4 T cells. Bulk CD4 T cells were purified from human PBMCs by magnetic bead selection. Cells were labeled with CFSE, and stimulated for 5 days with 1, 2.5 or 5ug/ml of anti-CD3 co-coated with 0, 2.5, 5, or 10ug/ml of human (a and c) or mouse (b and d) VISTA-Ig. Control-Ig was added to equalize the amount of fusion protein. (a and b) Overlays showing representative CFSE profiles. Cells are gated on live, singlet CD4 positive cells. (c and d) Percentage CFSE low cells are shown as mean +/- SD. *p

PDF file - 77KB, VISTA does not affect B cell proliferation. CD19 positive B cells were purified from human PBMCs by magnetic bead negative selection. (a and b) cells were stimulated in the presence of VISTA-Ig (a) or control Ig (b) at 5ug/ml for 4 days with 250ng/ml of CD40 agonist. Plots show representative CFSE profiles gated on live CD19 positive events. C) percent proliferation in the presence of decreasing concentrations of agonist in the presence of VISTA-Ig (filled bars) or control Ig (open bars) at 5ug/ml for 4 days. Data is representative of 3 separate experiments.

MicroRNAs (miRNAs) are a new class of short noncoding regulatory RNAs (18–25 nucleotides) that are involved in diverse developmental and pathologic processes. Altered miRNA expression has been associated with several types of human cancer. However, most studies did not establish whether miRNA expression changes occurred within cells undergoing malignant transformation. To obtain insight into miRNA deregulation in breast cancer, we implemented an in situ hybridization (ISH) method to reveal the spatial distribution of miRNA expression in archived formalin-fixed, paraffin-embedded specimens representing normal and tumor tissue from >100 patient cases. Here, we report that expression of miR-145 and miR-205 was restricted to the myoepithelial/basal cell compartment of normal mammary ducts and lobules, whereas their accumulation was reduced or completely eliminated in matching tumor specimens. Conversely, expression of other miRNAs was detected at varying levels predominantly within luminal epithelial cells in normal tissue; expression of miR-21 was frequently increased, whereas that of let-7a was decreased in malignant cells. We also analyzed the association of miRNA expression with that of epithelial markers; prognostic indicators such as estrogen receptor, progesterone receptor, and HER2; as well as clinical outcome data. This ISH approach provides a more direct and informative assessment of how altered miRNA expression contributes to breast carcinogenesis compared with miRNA expression profiling in gross tissue biopsies. Most significantly, early manifestation of altered miR-145 expression in atypical hyperplasia and carcinoma in situ lesions suggests that this miRNA may have a potential clinical application as a novel biomarker for early detection. [Cancer Res 2007;67(24):11612–20]

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Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer with an overall 5-year survival rate of less than 10%. The 1-year survival rate of patients with locally advanced or metastatic disease is abysmal. The aggressive nature of cancer cells, hypovascularization, extensive desmoplastic stroma, and immunosuppressive tumor microenvironment (TME) endows PDAC tumors with multiple mechanisms of drug resistance. With no obvious genetic mutation(s) driving tumor progression or metastatic transition, the challenges for understanding the biological mechanism(s) of these processes are paramount. A better understanding of the molecular and cellular mechanisms of these processes could lead to new diagnostic tools for patient management and new targets for therapeutic intervention. microRNAs (miRNAs) are an evolutionarily conserved gene class of short non-coding regulatory RNAs. miRNAs are an extensive regulatory layer that controls gene expression at the posttranscriptional level. This review focuses on preclinical models that functionally dissect miRNA activity in tumor progression or metastatic processes in PDAC. Collectively, these studies suggest an influence of miRNAs and RNA-RNA networks in the processes of epithelial to mesenchymal cell transition and cancer cell stemness. At a cell-type level, some miRNAs mainly influence cancer cell–intrinsic processes and pathways, whereas other miRNAs predominantly act in distinct cellular compartments of the TME to regulate fibroblast and immune cell functions and/or influence other cell types’ function via cell-to-cell communications by transfer of extracellular vesicles. At a molecular level, the influence of miRNA-mediated regulation often converges in core signaling pathways, including TGF-β, JAK/STAT, PI3K/AKT, and NF-κB.

Breast cancer (BC) is the leading cancer diagnosis in women. While many BC treatment options exist, only two methods are approved for prevention. Prophylactic mastectomy is an invasive surgery that removes the breast, thereby removing the targeted epithelial ductal tree cells, where most BC arises. Despite a 90% risk reduction, recovery time, risk of infection, pain management, and mental health toll on women deter them from this surgery. Hormonal preventatives, tamoxifen and raloxifene, reduce BC risk by up to 50%. However, increased risk of uterine cancer and intolerable sides effect deter women from this option. Therefore, new preventative methods are needed to maintain BC risk reduction while minimizing current BC prevention deterrents. Intraductal (ID) injection is a technique that can circumvent systemic toxicity by inserting a needle directly into the ductal tree opening. We study BC prevention using 70% ethanol (EtOH) as a cell killing, ablative solution for ID injections and have shown its effectiveness of ablation in an aggressive genetically engineered mouse model with minimum collateral tissue damage. EtOH is a safe, inexpensive reagent clinically used as a sclerosing and ablative agent. We introduced ethyl cellulose (EC), a gelling agent which further minimized damage outside the ductal tree. To confirm successful ductal tree filling without animal sacrifice, we introduced Tantalum Oxide (TaOx), a nanoparticle-based contrast agent for real time and long-term visualization of ductal tree filling. TaOx showed remarkable local retention, without impeding 70% EtOH ablative effects or EC compared to other contrast agents. Despite our refined ablative solution, ID injection is a difficult technique to perform with limited ability to confirm success of a fully filled ductal tree. To elucidate any impact of partial ductal tree filling, we utilized a Methyl-N-Nitrosourea (MNU) rat model. Rat models improve scalability toward clinical trials and increase ductal tree filling volume and control to study the impact of partial ductal tree filling. Our MNU rat model provides a time frame between carcinogen exposure and tumor formation optimal for prevention studies. Two weeks after intraperitoneal MNU injections, rats received full or half volume (partial) injections, fat pad (partial), or no injection into individual mammary glands. Rats were monitored for tumor formation from 2 weeks post injection until reaching euthanasia criteria. Fully injected glands had the highest tumor latency increase by 1 month (130 days, p < 0.0001) compared to non-injected controls (95 days). Partial and fat pad tumor latency also improved compared to control (122 days, p Citation Format: Erin K. Zaluzec, Mohamed Ashry, Elizabeth Kenyon, Elizabeth G. Phelps, Legend Kenney, Katherine Powell, Maximilian Volk, Shatadru Chakravarty, Jeremy M.L. Hix, Matti Kiupel, Erik Shapiro, Lorenzo F. Sempere. Impact of 70% ethanol intraductal injections in MNU rat models for breast cancer prevention. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4247.

Breast cancer (BC) is the most prevalent cancer and the second-leading cause of cancer-related death for women in the USA. For moderate-risk and, especially, low-risk women with a 1-in-8 lifelong chance of developing BC, there are very few options available to reduce their risk. For high-risk women, prophylactic mastectomy is currently the most effective procedure for preventing BC. Prophylactic mastectomy completely removes the mammary epithelial cells from which BC arises along with surrounding tissue. This aggressive surgical procedure affects women physically, emotionally, psychologically, aesthetically, and socially. Therefore, there is a need to develop new strategies for primary prevention that focus on high-risk women, but that, at the same time, could be also applied to moderate- and low-risk women. Our overall hypothesis is that the local killing of mammary epithelial cells will be as effective as prophylactic mastectomy in preventing BC, but with minimal side effects. Preclinical and clinical research studies as well as clinical application of intraductal (ID) procedures such as ductography for diagnostic imaging strongly support the translational feasibility of our approach. EtOH has been long used clinically as an ablative or sclerosing agent for local treatment. We recently showed that ID injection of a 70% EtOH solution is effective at locally ablating mammary epithelial cells with limited collateral tissue damage and at preventing tumor formation in an aggressive and multifocal mouse model of BC. We also recently developed tantalum oxide (TaOx) nanoparticles as a larger and higher radiopaque contrast agent with a much lower rate of outward diffusion than iodine-based contrast agents used in clinical ductography. Here, we investigated a refined EtOH formulation that consists of the addition of: i) TaOx nanoparticles as a high-resolution contrast agent to monitor in vivo filling of ductal trees and ablative effects of EtOH by computed tomography imaging; and ii) ethyl-cellulose (EC) as gelling agent to further minimize collateral tissue damage. This TaOx-based imaging approach provided unprecedented resolution to visualize individual ducts and branches of the ductal tree network after initial ID injection and architectural changes over 7 d due to local retention of TaOx nanoparticles in both PBS and EtOH solutions. EC is used clinically to improve delivery of EtOH and to limit EtOH diffusion from the intended target area in the treatment of venous malformations. ID injection of 70% EtOH/3% EC in nontransgenic mice and rats achieved the same epithelial ablation rate as 70% EtOH alone, while significantly minimizing collateral tissue damage. These results demonstrate the compatibility, safety, and stability of this refined EtOH formulation and support its further investigation in cancer-prone rodent and larger animal models as an innovative primary intervention strategy for BC prevention. Citation Format: Lorenzo Sempere, Elizabeth Kenyon, Shatadru Chakravarty, Maximilian Volk, Jeremy ML Hix, Erin Zaluzec, Matti Kiupel, Erik Shapiro. Image-guided intraductal ablation with refined ethanol solution for primary prevention of breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS8-33.

Breast cancer is the most prevalent cancer and the second-leading cause of cancer-related death for women in the USA. For high-risk women, prophylactic mastectomy is the most effective primary prevention strategy. Prophylactic mastectomy is an aggressive surgical procedure that completely removes the mammary epithelial cells from which breast cancer arises along with the surrounding tissue. We seek to develop a minimally invasive intraductal procedure as an alternative to prophylactic mastectomy to locally ablate the mammary epithelial cells before they can become malignant. We and others have developed an intraductal delivery procedure to reach and treat these epithelial cells in rodent models of breast cancer. While the mouse mammary gland with a single non-anastomosed ductal tree opening at the nipple has a much less complex and tortuous architecture than the human breast, chemically induced and genetically engineered mouse models of breast cancer are valuable to produce proof-of-concept studies of new preventative strategies. Here, we describe a procedure for intraductal delivery of an ethanol-based ablative solution containing micro-CT/X-ray tantalum-based contrast agent within the mouse mammary ductal tree for the therapeutic purpose of primary prevention of breast cancer. Intraductal delivery of aqueous reagents (e.g., cytotoxic compounds, siRNAs, AdCre) has been previously described in mouse models. Thus, we focus our protocol description on methodological modifications and unique experimental considerations for optimizing delivery of ethanol, for minimizing local and systemic side effects of ethanol administration, and for in vivo visualization of ductal tree filling via micro-CT/fluoroscopy imaging. Visualization of the ductal tree immediately after injection of a contrast-containing solution allows for confirmation of complete filling or unsuccessful outcomes such as underfilling or overfilling. This procedure can be applied for delivery and imaging of other ablative compounds aimed at either preventing tumor formation or locally treating early-stage tumors accessible via the ductal tree.

The microenvironment of pancreatic cancer adenocarcinoma (PDAC) is highly desmoplastic with distinct tumor-restraining and tumor-promoting fibroblast subpopulations. Re-education rather than indiscriminate elimination of these fibroblasts has emerged as a new strategy for combination therapy. Here, we studied the effects of global loss of profibrotic noncoding regulatory microRNA-21 (miR-21) in K-Ras-driven p53-deleted genetically engineered mouse models of PDAC. Strikingly, loss of miR-21 accelerated tumor initiation via mucinous cystic neoplastic lesions and progression to locally advanced invasive carcinoma from which animals precipitously succumbed at an early age. The absence of tumor-restraining myofibroblasts and a massive infiltrate of immune cells were salient phenotypic features of global miR-21 loss. Stromal miR-21 activity was required for induction of tumor-restraining myofibroblasts in in vivo isograft transplantation experiments. Low miR-21 expression negatively correlated with a fibroblast gene expression signature and positively with an immune cell gene expression signature in The Cancer Genome Atlas PDAC data set (n = 156) mirroring findings in the mouse models. Our results exposed an overall tumor-suppressive function of miR-21 in in vivo PDAC models. These results have important clinical implications for anti-miR-21-based inhibitory therapeutic approaches under consideration for PDAC and other cancer types. Mechanistic dissection of the cell-intrinsic role of miR-21 in cancer-associated fibroblasts and other cell types will be needed to inform best strategies for pharmacological modulation of miR-21 activity to remodel the tumor microenvironment and enhance treatment response in PDAC.

microRNAs are short noncoding RNAs that regulate protein expression posttranscriptionally. We previously showed that miR-155 promotes effector CD8+ T-cell responses. However, little is known about the regulation of miR-155 expression. Here, we report that antigen affinity and dose determine miR-155 expression in CD8+ T cells. In B16 tumors expressing a low-affinity antigen ligand, tumor-specific infiltrating CD8+ T cells showed variable miR-155 expression, whereby high miR-155 expression was associated with more cytokine-producing cells and tumor control. Moreover, anti–PD-1 treatment led to both increased miR-155 expression and tumor control by specific CD8+ T cells. In addition, miR-155 overexpression enhanced exhausted CD8+ T-cell persistence in the LCMV cl13 chronic viral infection model. In agreement with these observations in mouse models, miR-155 expression in human effector memory CD8+ T cells positively correlated with their frequencies in tumor-infiltrated lymph nodes of melanoma patients. Low miR-155 target gene signature in tumors was associated with prolonged overall survival in melanoma patients. Altogether, these results raise the possibility that high miR-155 expression in CD8+ tumor-infiltrating T cells may be a surrogate marker of the relative potency of in situ antigen-specific CD8+ T-cell responses.

It has been almost two decades since the first link between microRNAs and cancer was established. In the ensuing years, this abundant class of short noncoding regulatory RNAs has been studied in virtually all cancer types. This tremendously large body of research has generated innovative technological advances for detection of microRNAs in tissue and bodily fluids, identified the diagnostic, prognostic, and/or predictive value of individual microRNAs or microRNA signatures as potential biomarkers for patient management, shed light on regulatory mechanisms of RNA–RNA interactions that modulate gene expression, uncovered cell‐autonomous and cell‐to‐cell communication roles of specific microRNAs, and developed a battery of viral and nonviral delivery approaches for therapeutic intervention. Despite these intense and prolific research efforts in preclinical and clinical settings, there are a limited number of microRNA‐based applications that have been incorporated into clinical practice. We review recent literature and ongoing clinical trials that highlight most promising approaches and standing challenges to translate these findings into viable microRNA‐based clinical tools for cancer medicine. This article is categorized under:RNA in Disease and Development > RNA in Disease, microRNAs are short noncoding regulatory RNAs with important roles in cancer biology. microRNAs are informative biomarkers for early cancer detection and treatment decisions. microRNAs are being evaluated in clinical trials as new drugs for personalized cancer medicine.

Pancreatic ductal adenocarcinoma (PDAC) originates from both ductal and acinar cells of the pancreas. The highly abundant and acinar cell-enriched miR-216a is reduced during in vitro acinar ductal metaplasia (ADM), throughout PanIN progression, during the development of pancreatitis in mice and humans, and during development of mouse and human PDAC. To investigate the contribution of miR-216a in the development of PDAC, we generated a miR-216a germline knockout mouse (216aKO) via CRISPR genetic editing of a minimal and precise deletion of the miR-216a precursor sequence without affecting the host gene. We crossed this 216aKO mouse to the LSL-KrasG12D; LSL-Trp53Flox/+; Pdx1Cre/+ (KPC) mice to produce a transgenic mouse with an activating Kras mutation, p53 deletion, and knockout of miR-216a (referred to here as miR216aKPC). miR216aKPC displayed an increase in tumor progression compared to KPC and had reduced survival - median 15 weeks miR216aKPC vs. 25 weeks for KPC. miR216aKPC produced lung metastasis by 12 weeks of age which were not present in the lungs of similarly aged KPC mice. Transfection of miR-216a mimetic oligo into cell lines derived from KPC or 216aKPC mice did not reduce viability or alter cellular morphology, suggesting that a potential tumor suppressive role of miR-216a functions during the early stages of PDAC development. A three dimensional ADM assay using acinar cells derived from both mouse and human pancreata will be applied to investigate the contributions of miR-216a on the development of ADM and cell polarity. To discover miR-216a target genes that are responsible for the increased tumorigenesis, cell lines derived from miR216aKPC will be transfected with miR-216a mimetic or scrambled control oligo followed by RNA sequencing. Our results thus far suggest a tumor suppressive role for miR-216a in the early development of PDAC and future studies will investigate molecular and cellular mechanisms driving acinar cell-induced PDAC. Citation Format: Andrew A. Brock, Katherine Powell, Thomas D. Schmittgen, Lorenzo F. Sempere. Enhanced tumorigenesis in a novel miR-216a knockout/KPC mouse model of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5821.

microRNAs are an important class of noncoding regulatory RNAs with functional roles in development, physiology, and disease. Visualization of microRNA expression at a single-cell level has contributed to a better understanding of their biological function in animal models and their etiological contribution to human diseases. In addition, several microRNAs have been highlighted as potential biomarkers carrying diagnostic and prognostic information. Co-detection of microRNA expression with that of cell-type-specific proteins can enhance the interpretative power of expression changes during development or altered expression in pathological conditions. Here, we describe an automated fluorescence-based five-color multiplex assay for co-detection of microRNA (e.g., miR-10b, miR-21, miR-205), noncoding RNA (e.g., snRNA U6, 18S rRNA), and protein expression (e.g., cytokeratin 19, vimentin, collagen I) in paraffin-embedded formalin-fixed tissue slides on a Leica Bond Rx staining station. While this protocol uses mainly mouse tissues to demonstrate multiplex detection, it can be generally applied to single-cell expression analysis of other animal models and clinical specimens.

Here, we describe the synthesis, characterization and in vitro and in vivo performance of a series of tantalum oxide (TaO(x)) based nanoparticles (NPs) for computed tomography (CT). Five distinct versions of 9–12 nm diameter silane coated TaO(x) nanocrystals (NCs) were fabricated by a sol-gel method with varying degrees of hydrophilicity and with or without fluorescence, with the highest reported Ta content to date (78%). Highly hydrophilic NCs were left bare and were evaluated in vivo in mice for micro-CT of full body vasculature, where following intravenous injection, TaO(x) NCs demonstrate high vascular CT contrast, circulation in blood for ~ 3 h, and eventual accumulation in RES organs; and following injection locally in the mammary gland, where the full ductal tree structure can be clearly delineated. Partially hydrophilic NCs were encapsulated within mesoporous silica nanoparticles (MSNPs; TaO(x)@MSNPs) and hydrophobic NCs were encapsulated within poly(lactic-co-glycolic acid) (PLGA; TaO(x)@PLGA) NPs, serving as potential CT-imagable drug delivery vehicles. Bolus intramuscular injections of TaO(x)@PLGA NPs and TaO(x)@MSNPs to mimic the accumulation of NPs at a tumor site produce high signal enhancement in mice. In vitro studies on bare NCs and formuated NPs demonstrate high cytocompatibility and low dissolution of TaO(x). This work solidifies that TaO(x)-based NPs are versatile contrast agents for CT.

microRNAs are an important class of noncoding regulatory RNAs with functional roles in development, physiology, and disease. Visualization of microRNA expression at a single-cell level has contributed to a better understanding of their biological function in animal models and their etiological contribution to human diseases. In addition, several microRNAs have been highlighted as potential biomarkers carrying diagnostic and prognostic information. Co-detection of microRNA expression with that of cell-type-specific proteins can enhance the interpretative power of expression changes during development or altered expression in pathological conditions. Here, we describe an automated fluorescence-based five-color multiplex assay for co-detection of microRNA (e.g., miR-10b, miR-21, miR-205), noncoding RNA (e.g., snRNA U6, 18S rRNA), and protein expression (e.g., cytokeratin 19, vimentin, collagen I) in paraffin-embedded formalin-fixed tissue slides on a Leica Bond Rx staining station. While this protocol uses mainly mouse tissues to demonstrate multiplex detection, it can be generally applied to single-cell expression analysis of other animal models and clinical specimens.

Traditional targeted therapeutic agents have relied on small synthetic molecules or large proteins, such as monoclonal antibodies. These agents leave a lot of therapeutic targets undruggable because of the lack or inaccessibility of active sites and/or pockets in their three-dimensional structure that can be chemically engaged. RNA presents an attractive, transformative opportunity to reach any genetic target with therapeutic intent. RNA therapeutic design is amenable to modularity and tunability and is based on a computational blueprint presented by the genetic code. Here, we will focus on short non-coding RNAs (sncRNAs) as a promising therapeutic modality because of their potency and versatility. We review recent progress towards clinical application of small interfering RNAs (siRNAs) for single-target therapy and microRNA (miRNA) activity modulators for multi-target therapy. siRNAs derive their potency from the fact that the underlying RNA interference (RNAi) mechanism is catalytic and reliant on post-transcriptional mRNA degradation. Therapeutic siRNAs can be designed against virtually any mRNA sequence in the transcriptome and specifically target a disease-causing mRNA variant. Two main classes of microRNA activity modulators exist to increase (miRNA mimics) or decrease (anti-miRNA inhibitors) the function of a specific microRNA. Since a single microRNA regulates the expression of multiple target genes, a miRNA activity modulator can have a more profound effect on global gene expression and protein output than siRNAs do. Both types of sncRNA-based drugs have been investigated in clinical trials and some siRNAs have already been granted FDA approval for the treatment of genetic, cardiometabolic, and infectious diseases. Here, we detail clinical results using siRNA and miRNA therapeutics and present an outlook for the potential of these sncRNAs in medicine.

Background Prophylactic mastectomy is the most effective intervention to prevent breast cancer. However, this major surgery has life-changing consequences at the physical, emotional, psychological, and social levels. Therefore, only high-risk individuals consider this aggressive procedure, which completely removes the mammary epithelial cells from which breast cancer arises along with surrounding tissue. Here, we seek to develop a minimally invasive procedure as an alternative to prophylactic mastectomy by intraductal (ID) delivery of a cell-killing solution that locally ablates the mammary epithelial cells before they become malignant. Methods After ID injection of a 70% ethanol-containing solution in FVB/NJ female animals, ex vivo dual stained whole-mount tissue analysis and in vivo X-ray microcomputed tomography imaging were used to visualize ductal tree filling, and histological and multiplex immunohistochemical assays were used to characterize ablative effects and quantitate the number of intact epithelial cells and stroma. After ID injection of 70% ethanol or other solutions in cancer-prone FVB-Tg-C3(1)-TAg female animals, mammary glands were palpated weekly to establish tumor latency and examined after necropsy to record tumor incidence. Statistical difference in median tumor latency and tumor incidence between experimental groups was analyzed by log-rank test and logistic mixed-effects model, respectively. Results We report that ID injection of 70% ethanol effectively ablates the mammary epithelia with limited collateral damage to surrounding stroma and vasculature in the murine ductal tree. ID injection of 70% ethanol into the mammary glands of the C3(1)-TAg multifocal breast cancer model significantly delayed tumor formation (median latency of 150 days in the untreated control group [n = 25] vs. 217 days in the ethanol-treated group [n = 13], p value p value Conclusions This preclinical study demonstrates the feasibility of local ductal tree ablation as a novel strategy for primary prevention of breast cancer. Given the existing clinical uses of ethanol, ethanol-based ablation protocols could be readily implemented in first-in-human clinical trials for high-risk individuals.

In 1993, the Ambros lab reported the cloning and developmental function of lin-4, the first microRNA [...]

Light provides the primary timing signal that enables fine-tuned behavioral and hormonal entrainment of circadian rhythms to the environment. Light is transmitted from the eye to the brain through the retinohypothalamic tract, where one target is the hypothalamic suprachiasmatic nucleus (SCN), which generates self-sustained circadian rhythms. The vasoactive intestinal polypeptide (VIP) expressing neurons of the SCN relay light information to peripheral cells and tissues through control of hormonal and nervous signals, allowing synchronization of molecular clocks located in individual cells throughout the body. Non-natural light cycles, ie shiftwork, and weakened SCN function through genetic manipulation, disrupt the body’s circadian rhythms, causing deregulated hormone release, metabolic disorders, and negative effects on reproductive systems such as irregular menstrual cycles and decreased sperm count. To further our understanding of how the SCN translates light information into neuroendocrine control of fertility, we conditionally deleted the SCN enriched transcription factor Ventral anterior homeobox 1 (Vax1) in post-developmental VIP neurons, generating Vax1-flox/flox:Vip-Cre+ (cKO) mice. To determine if the SCN timekeeping function was impacted in cKO mice, we single housed males and females with running wheels to examine activity during both 12-hour light/dark cycles and in constant darkness. Wheel-running behavior in constant darkness revealed a shortening of the endogenous free-running period (Tau) of the SCN. Aside from Tau, wheel running behaviors were comparable to controls. Weakened SCN output can negatively impact fertility. While on 12-hour light/dark cycles, we found a modest, but significant change in follicle stimulating hormone and estrogen in cKO females and a reduced sensitivity of GnRH neurons to kisspeptin in males. The changes in hormone release were associated with a slightly lengthened estrous cycle in cKO females and reduced sperm quality in cKO males. To identify the molecular origin of the shortened SCN period, we used immunohistochemistry and RNAscope to examine expression of Vip. We found that diestrus cKO females had a significant reduction in Vip expression at ZT16 and preliminary data suggest a reduction in the circadian clock gene Bmal1. Together, these studies identify a novel role of VAX1 in VIP neurons where VAX1 is required for VIP expression and circadian timekeeping. Loss of VAX1 in VIP neurons weakens SCN output, deregulating reproductive hormone release and modestly reducing reproductive function in both males and females.

Pancreatic ductal adenocarcinoma (PDAC) is a recalcitrant and lethal disease with an overall 5-year survival rate of less than 8%. The abundance and heterogeneity of cancer-associated fibroblasts (CAFs) in a highly desmoplastic tumor microenvironment are unique features of PDAC. Therapeutic strategies aimed at indiscriminately eliminating CAFs have failed in clinical trials. The opposing tumor-restraining or tumor-promoting role of distinct CAFs subpopulations of has emerged as a new paradigm in PDAC basic and translational research. Understanding how the composition of these distinct CAF subpopulations is affected by and interferes with current treatments is a key question to solve in order to obtain more effective and durable clinical responses. Genetic studies in K-Ras-driven genetically engineered mouse models (GEMMs) indicate that α-smooth muscle actin-expressing (SMA+) CAFs have a predominant tumor-restraining role. Here, we studied global loss of pro-fibrotic non-coding regulatory microRNA-21 (miR-21) in PDAC GEMMs. We generated mouse strains carrying wild type or knockout alleles of Mir-21 in a well-characterized K-Ras-driven, p53-deleted PDAC model (LSL-KrasG12D; p53lox/+;Pdx1-Cre, KPC). Even though miR-21 expression was upregulated in cancer cells and cancer-associated fibroblasts in KPC tumors, global loss of miR-21 activity did not inhibit tumor growth. Strikingly, loss of miR-21 accelerated tumor initiation via mucinous cystic neoplastic lesions and progression to locally advanced invasive carcinoma from which animals precipitously succumbed at an early age. We uncovered a cell-autonomous requirement of miR-21 activity for induction of tumor-restraining SMA+ CAFs, whose absence led to a profound remodeling of the stroma and massive infiltrate of tumor-promoting immune cells. Low miR-21 expression negatively correlated with a fibroblast gene expression signature and positively with an immune cell gene expression signature in TCGA PDAC data set (n = 156) mirroring findings in GEMMs. In vitro and in vivo isograft experiments showed that miR-21 activity was dispensable for cancer cell growth, but required for complete execution of TGF-β-mediated programs. Our results suggest that modulation of miR-21 activity may provide a novel therapeutic opportunity to affect the composition of CAF subpopulations. We will present our genetic and pharmacological approaches to modulate stromal activity of miR-21 in established disease in order to enhance current chemotherapy and/or immunotherapy treatments. Citation Format: Josh Schipper, Brooke Jackson, Ian Beddows, Elizabeth Kenyon, Katie Powell, Neil Robertson, Galen Hostetter, Matti Kiupel, Anna Moore, Jose Conejo-Garcia, Lorenzo sempere. Tumor-suppressive stromal activity of pro-fibrogenic microRNA-21 in initiation and progression of K-Ras-driven mouse models of pancreatic cancer [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 5719.

Certain experiments involve the high-throughput quantification of image data, thus requiring algorithms for automation. A challenge in the development of such algorithms is to properly interpret signals over a broad range of image characteristics, without the need for manual adjustment of parameters. Here we present a new approach for locating signals in image data, called Segment and Fit Thresholding (SFT). The method assesses statistical characteristics of small segments of the image and determines the best-fit trends between the statistics. Based on the relationships, SFT identifies segments belonging to background regions; analyzes the background to determine optimal thresholds; and analyzes all segments to identify signal pixels. We optimized the initial settings for locating background and signal in antibody microarray and immunofluorescence data and found that SFT performed well over multiple, diverse image characteristics without readjustment of settings. When used for the automated analysis of multi-color, tissue-microarray images, SFT correctly found the overlap of markers with known subcellular localization, and it performed better than a fixed threshold and Otsu’s method for selected images. SFT promises to advance the goal of full automation in image analysis.

Bladder cancer is the fourth most common cancer among men in the United States and more than half of patients experience recurrences within 5 years after initial diagnosis. Additional clinically informative and actionable biomarkers of the recurrent bladder cancer phenotypes are needed to improve screening and molecular therapeutic approaches for recurrence prevention. MicroRNA-34a (miR-34a) is a short noncoding regulatory RNA with tumor suppressive attributes. We leveraged our unique, large, population-based prognostic study of bladder cancer in New Hampshire, United States to evaluate miR-34a expression levels in individual tumor cells to assess prognostic value. We collected detailed exposure and medical history data, as well as tumor tissue specimens from bladder patients and followed them long-term for recurrence, progression and survival. Fluorescence-based in situ hybridization assays were performed on urothelial carcinoma tissue specimens (n = 229). A larger proportion of the nonmuscle invasive tumors had high levels of miR-34a within the carcinoma cells compared to those tumors that were muscle invasive. Patients with high miR-34a levels in their baseline nonmuscle invasive tumors experienced lower risks of recurrence (adjusted hazard ratio 0.57, 95% confidence interval 0.34-0.93). Consistent with these observations, we demonstrated a functional tumor suppressive role for miR-34a in cultured urothelial cells, including reduced matrigel invasion and growth in soft agar. Our results highlight the need for further clinical studies of miR-34a as a guide for recurrence screening and as a possible candidate therapeutic target in the bladder.

Soon after the discovery of microRNAs over 15 years ago, a myriad of research groups around the world sought to develop clinical applications in breast cancer for these short, noncoding, regulatory RNAs. While little of this knowledge has translated into the clinic, the recent research explosion on cell-to-cell communication via exosomes and other extracellular vesicles has rekindled interest in microRNA-based clinical applications. microRNAs appear to be a preferential and important cargo of exosomes in mediating biological effects in recipient cells. This review highlights recent studies on the biology of exosomal microRNAs (exo-miRNAs) and discusses potential clinical applications. From a diagnostic perspective, circulating exo-miRNAs may represent breast cancer cell content and/or tumor microenvironmental reactions to cancer cell growth. Thus, serum or plasma analysis of exo-miRNAs could be useful for early disease detection or for monitoring treatment response and disease progression. From a therapeutic perspective, exo-miRNAs derived from different cell types have been implicated in supporting or restraining tumor growth, conferring drug resistance, and preparing the metastatic niche. Strategies to interfere with the loading or delivery of tumor-promoting exo-miRNAs or to replenish tumor-suppressive miRNAs via exosomal delivery are under investigation. These recent studies provide new hope and opportunities, but study design limitations and technical challenges will need to be overcome before seriously considering clinical application of exo-miRNAs.

miRNAs are short, non-coding, regulatory RNAs that exert cell type-dependent, context-dependent, transcriptome-wide gene expression control under physiological and pathological conditions. Tissue slide-based assays provide qualitative (tumor compartment) and semi-quantitative (expression levels) information about altered miRNA expression at single-cell resolution in clinical tumor specimens. Reviewed here are key technological advances in the last 5 years that have led to implementation of fully automated, robust and reproducible tissue slide-based assays for in situ miRNA detection on US FDA-approved instruments; recent tissue slide-based discovery studies that suggest potential clinical applications of specific miRNAs in cancer medicine are highlighted; and the challenges in bringing tissue slide-based miRNA assays into the clinic are discussed, including clinical validation, biomarker performance, biomarker space and integration with other biomarkers.

The tumor microenvironment of pancreatic cancer adenocarcinoma (PDAC) is highly desmoplastic with distinct tumor-restraining and tumor-promoting fibroblast subpopulations. Re-education rather than indiscriminate elimination of these fibroblasts has emerged as a new strategy for combination therapy. Here, we studied global loss of profibrotic noncoding regulatory microRNA-21 (miR-21) in K-Ras-driven genetically engineered mouse models of PDAC. We generated mouse strains carrying wild-type or knockout alleles of Mir-21 in a well-characterized K-Ras-driven, p53-deleted PDAC model (LSL-KrasG12D; p53lox/+;Pdx1-Cre, KPC). Even though miR-21 expression was upregulated in cancer cells and cancer-associated fibroblasts in KPC tumors, global loss of miR-21 activity did not inhibit tumor growth. Strikingly, loss of miR-21 accelerated tumor initiation via mucinous cystic neoplastic lesions and progression to locally advanced invasive carcinoma from which animals precipitously succumbed at an early age. We uncovered a cell-autonomous requirement of miR-21 activity for induction of tumor-restraining myofibroblasts, whose absence led to a profound remodeling of the stroma and massive infiltrate of tumor-permissive immune cells. Low miR-21 expression negatively correlated with a fibroblast gene expression signature and positively with an immune cell gene expression signature in TCGA PDAC data set (n = 156) mirroring findings in genetic models. In vitro and in vivo isograft experiments showed that miR-21 activity was dispensable for cancer cell growth but required for complete execution of TGF-b-mediated programs. Our results suggest that modulation of miR-21 activity may provide a novel therapeutic opportunity to affect the composition of fibroblast subpopulations and enhance current chemotherapy and/or immunotherapy treatments. Citation Format: Josh Schipper, Ian Beddows, Matti Kiupel, Jose Conejo-Garcia, Lorenzo Sempere. Genetic ablation of microRNA-21 profoundly remodels stroma and shortens survival of K-Ras-driven pancreatic cancer mouse models [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A48.

Modulating the activity of miRNAs provides opportunities for novel cancer interventions. However, low bioavailability and poor cellular uptake are major challenges for delivering miRNA mimetics specifically to tumor cells. Here, we took advantage of the spontaneous enhanced endocytic activity of ovarian cancer-associated dendritic cells (DC) to selectively supplement the immunostimulatory miRNA miR-155. In vivo processing of nanoparticles carrying oligonucleotide duplexes mimicking the bulged structure of endogenous pre-miRNA (but not siRNA-like oligonucleotides) dramatically augmented miR-155 activity without saturating the RNA-induced silencing complex. Endogenous processing of synthetic miR-155 favored Ago2 and, to a lesser extent, Ago4 loading, resulting in genome-wide transcriptional changes that included silencing of multiple immunosuppressive mediators. Correspondingly, tumor-infiltrating DCs were transformed from immunosuppressive to highly immunostimulatory cells capable of triggering potent antitumor responses that abrogated the progression of established ovarian cancers. Our results show both the feasibility and therapeutic potential of supplementing/replenishing miRNAs in vivo using nonviral approaches to boost protective immunity against lethal tumors. Thus, we provide a platform, an optimized design, and a mechanistic rationale for the clinical testing of nonviral miRNA mimetics. Cancer Res; 72(7); 1683–93. ©2012 AACR.

The promise of personalized medicine is highly dependent on the identification of biomarkers that inform diagnostic decisions and treatment options, as well as on the accurate, rapid and cost-effective detection and interpretation of these biomarkers. miRNAs, which are short noncoding regulatory RNAs, are rapidly emerging as a novel class of biomarkers with a unique set of biological and chemical properties that makes them very appealing candidates for theranostic applications in cancer. Since the utility of some protein-encoding gene biomarkers is already exploited in routine clinical practice, it will be important to identify areas in which miRNAs provide complementary or superior information to these existing (and other translational) biomarkers to enhance the diagnostic, prognostic and predictive power of molecular characterization of tumors. In this article, the challenges and opportunities for integration of miRNA-based assays in the clinical toolkit to improve care and management of patients afflicted with solid tumors will be discussed.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy. Diagnosis and management of PDAC are hampered by the absence of sensitive and specific disease biomarkers. MicroRNAs (miRNA) are noncoding regulatory RNAs involved in initiation and progression of human cancers. In this study, we sought to determine whether miR-10b could serve as a biomarker for PDAC. Experimental Design: miRNA expression was characterized by fluorescence-based in situ hybridization using locked nucleic acid–modified DNA probes against miR-10b, miR-21, miR-155, miR-196a, and miR-210, followed by codetection of proteins by immunohistochemistry on the same tissue sections. miRNA expression in surgically resected PDAC tissues and in endoscopic ultrasonography (EUS)-guided fine-needle aspirate (EUS-FNA) samples was analyzed in cytokeratin 19 (CK19)–positive epithelial cells using optical intensity analysis. Results: In 10 resected PDAC samples, miR-10b was the most frequently and consistently overexpressed miRNA among characterized miRNAs, exhibiting a four-fold increase in the cancer cells (P = 0.012). Given this preferential overexpression of miR-10b, we sought to determine whether miR-10b expression was clinically relevant. Accordingly, miR-10b expression was examined in 106 EUS-FNA samples obtained from pancreatic lesions. miR-10b expression was increased in cancer cells compared with CK19-positive epithelial cells in benign lesions (P = 0.0001). In patients with PDACs, lower levels of miR-10b were associated with improved response to multimodality neoadjuvant therapy, likelihood of surgical resection, delayed time to metastasis, and increased survival. Conclusion: miR-10b is a novel diagnostic biomarker for PDACs when assessing pancreatic lesions. Expression of miR-10b is predictive of response to neoadjuvant therapy and outcome in this disease. Clin Cancer Res; 17(17); 5812–21. ©2011 AACR.

Transforming Growth Factor-β (TGFβ) exerts cell type-specific and context-dependent effects. Understanding the intrinsic actions of TGFβ on cancer cells in pancreatic ductal adenocarcinoma (PDAC) is a prerequisite for rationalizing clinical implementation of TGFβ targeted therapies. Since the tumor microenvironment can affect how cancer cells respond to TGFβ, we employed a novel 3-dimensional (3D) culturing system to recapitulate stromal and extracellular matrix interactions. We show here that TGFβ stimulates the growth of several human and murine pancreatic cancer cell lines (PCCs) when embedded in a 3% collagen IV/laminin-rich gelatinous medium (Matrigel™) over a solidified layer of soft agar. Moreover, in this novel 3D model, concomitant treatment with TGFβ1 and epidermal growth factor (EGF) enhanced PCC growth to a greater extent than either growth factor alone and conferred increased chemoresistance to cytotoxic compounds. These cooperative growth-stimulatory effects were blocked by pharmacological inhibition of either the TGFβ type I receptor with SB431542 or the EGF receptor with erlotinib. Co-incubation with SB431542 and erlotinib enhanced the efficacy of gemcitabine and cisplatin in PCCs and in primary cell cultures established from pancreata of genetically-engineered mouse models of PDAC. These findings suggest that concomitant inhibition of TGFβ and EGF signaling may represent an effective therapeutic strategy in PDAC, and that this 3D culturing system could be utilized to test ex vivo the therapeutic response of pancreatic tumor biopsies from PDAC patients, thereby providing a functional assay to facilitate personalized targeted therapies.

Background: Immune checkpoint blockade remarkably provides durable clinical response in solid tumors and immunohistochemistry (IHC) measures are current companion diagnostics. Currently four (4) IHC companion diagnostics of Programed Death Ligand (PD-L1) are components of FDA approved clinical trials. The marked clinical responses seen in checkpoint inhibition outpace basic science advances. Our previous reports of interferon mediated viral response against dsRNA intermediates, namely in colon and bladder cancer, have examined the role of global demethylation (5-AZA) and combination therapies. Materials and Methods: Cell lines HCT116 (colon) and T24 (bladder) were treated with single and combination therapy of 5-AZA and Vitamin C were formalin fixed and processed for IHC staining for Ki67, MHL1 and markers of viral response: dsRNA markers (K1, J2), interferon activation (RIG I, MDA5, IFIT1, IFI27, IRF7, IRF9, and pSTAT1. Immune related markers included CD4, CD8, CD68, CD163 and PD-L1 (clone SP263). TMA sections were stained by automated immunostainers to include a subset of patient-matched whole sections. IHC staining documented for protein abundance (intensity 0-3) and cellular localization (nuclear, cytoplasmic, membranous) Results: Colon cell line experiments confirmed viral response activation with correlative staining of dsRNA antibodies with viral response markers RIG1, MDA5, IFIT1, IFI27, IRF7 and pSTAT1 with robust staining in formalin fixed paraffin embedded (FFPE) tissues with the exception of IFI27 and RIG1. Immune and macrophage markers CD8, CD4, CD68, and CD163 showed variable correlation with viral response markers and PD-L1 staining heterogeneous and most prevalent in non-small cell lung cancer (34%). Of 3 MMR deficient colon cancers, 2 stained positive for PD-L1. MDA5 staining was present in tumor and stroma but stronger peri-tumoral suggesting relation to tumor infiltrating lymphocytes. TMA cores and corresponding whole sections with co-expressed PD-L1 and MDA5 showed marked diminished CD8 staining as verified by nuclear quantitative algorithm. Summary: Here we evaluate the viral response phenotype via global demethylation in solid tumors with emphasis on colon and bladder cancer. lung and breast cancer. IHC methods in cell lines and FFPE clinical tissues provide a robust strategy to link viral and immune response in clinical tissue samples and provides glimpses of viral and immune pathways that might better elucidate tumor biology and thereby predict tumor response to drug regimens that include checkpoint inhibitors. Citation Format: Galen Hostetter, Liu Minmin, Marie Mustert, Bree Berghuis, Lisa Turner, Peter Jones, Steve Baylin, Scott Jewell, Lorenzo Sempere. Viral response markers in immune-competent solid tumors by immunohistochemistry [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A39.

The role of innate and adaptive immunity in a wide range of human tumors has shown critical roles, though sometimes competing, in numerous immune components in both the tumor and the tumor microenvironment (TME). The PD-1/PD-L1 interaction in both tumor and the TME has become high priority in correlative biomarkers of human disease with increasing number of targeted agents showing high levels of durable response. The in situ analysis of viral defense pathway, interferon response, and PD-L1 status has provided strong preliminary data of correlative staining by immunohistochemistry in colon and lung carcinomas. Generally, such human tumors arise in organs with frequent and high exposure to external/foreign antigens. Here we expand our IHC staining to include human tumors arising from sites with varied immune activity to include uterine wall (leiomyosarcoma), subcutaneous connective tissue to include dermatofibrosarcoma protuberans (DFSP), soft tissue sarcoma, fibrosarcoma, and osteosarcoma. IHC panel includes immune markers of CD4, CD8, CD68 (M1 macrophages), CD163 (M2 macrophages); dsRNA antibodies (J2 and K1); MDA 5, RIG 1, STAT 1, and pSTAT1; PD-L1 and PD-1; and interferon response markers IRF 3, IRF 7, and IFIT 1. Immunostaining is ongoing and supports preliminary sarcoma phenotype of quite varied PD-L1 immunoreactivity with lower prevalence than lung and colon carcinomas. Immune markers of T-cell subset and macrophage M1 and M2 are significantly lower in sarcoma samples and detailed analysis of M1 and M2 macrophages in DFSP, STS, fibrosarcomas, and osteosarcoma. Each tumor type is represented on single TMA with 8-12 samples each from the Van Andel Institute Biorepository Core. In our experience, tissue site density and macrophage subtypes and difference in polarized macrophages (M2) in relation to CD4 and CD8 markers were significant in relation to PD-L1 and require further exploration. Improved characterization of PD-L1 pathway and potential surrogate markers to compile immune competency profiles will include early results from our IHC staining for dsRNA fragments to lend support to endogenous retroviral activation in a wide range of both carcinomas and sarcomas. Citation Format: Galen Hostetter, Lorenzo Sempere, Scott Jewell, Steve Baylin. Assessing macrophage polarization in sarcomas with PD-L1 correlates [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B33.