Mahipal Suraneni

The PSA−/lo Prostate Cancer Cell Population Harbors Self-Renewing Long-Term Tumor-Propagating Cells that Resist Castration

Prostate cancer (PCa) is heterogeneous and contains both differentiated and undifferentiated tumor cells, but the relative functional contribution of these two cell populations remains unclear. Here we report distinct molecular, cellular, and tumor-propagating properties of PCa cells that express high (PSA(+)) and low (PSA(-/lo)) levels of the differentiation marker PSA. PSA(-/lo) PCa cells are quiescent and refractory to stresses including androgen deprivation, exhibit high clonogenic potential, and possess long-term tumor-propagating capacity. They preferentially express stem cell genes and can undergo asymmetric cell division to generate PSA(+) cells. Importantly, PSA(-/lo) PCa cells can initiate robust tumor development and resist androgen ablation in castrated hosts, and they harbor highly tumorigenic castration-resistant PCa cells that can be prospectively enriched using ALDH(+)CD44(+)α2β1(+) phenotype. In contrast, PSA(+) PCa cells possess more limited tumor-propagating capacity, undergo symmetric division, and are sensitive to castration. Altogether, our study suggests that PSA(-/lo) cells may represent a critical source of castration-resistant PCa cells.

Critical and Distinct Roles of p16 and Telomerase in Regulating the Proliferative Life Span of Normal Human Prostate Epithelial Progenitor Cells

Normal human prostate (NHP) epithelial cells undergo senescence in vitro and in vivo, but the underlying molecular mechanisms remain obscure. Here we show that the senescence of primary NHP cells, which are immunophenotyped as intermediate basal-like cells expressing progenitor cell markers CD44, α2β1, p63, hTERT, and CK5/CK18, involves loss of telomerase expression, up-regulation of p16, and activation of p53. Using genetically defined manipulations of these three signaling pathways, we show that p16 is the primary determinant of the NHP cell proliferative capacity and that hTERT is required for unlimited proliferative life span. Hence, suppression of p16 significantly extends NHP cell life span, but both p16 inhibition and hTERT are required to immortalize NHP cells. Importantly, immortalized NHP cells retain expression of most progenitor markers, demonstrate gene expression profiles characteristic of proliferating progenitor cells, and possess multilineage differentiation potential generating functional prostatic glands. Our studies shed important light on the molecular mechanisms regulating the proliferative life span of NHP progenitor cells.

TRANSGENIC EXPRESSION OF 15-LIPOXYGENASE 2 (15-LOX2) IN MOUSE PROSTATE LEADS TO HYPERPLASIA AND CELL SENESCENCE

15-Lipoxygenase 2 (15-LOX2), a lipid-peroxidizing enzyme, is mainly expressed in the luminal compartment of the normal human prostate, and is often decreased or lost in prostate cancer. Previous studies from our lab implicate 15-LOX2 as a functional tumor suppressor. To better understand the biological role of 15-LOX2 in vivo, we generated prostate-specific 15-LOX2 transgenic mice using the ARR2PB promoter. Unexpectedly, transgenic expression of 15-LOX2 or 15-LOX2sv-b, a splice variant that lacks arachidonic acid-metabolizing activity, resulted in age-dependent prostatic hyperplasia and enlargement of the prostate. Prostatic hyperplasia induced by both 15-LOX2 and 15-LOX2sv-b was associated with an increase in luminal and Ki-67+ cells; however, 15-LOX2-transgenic prostates also showed a prominent increase in basal cells. Microarray analysis revealed distinct gene expression profiles that could help explain the prostate phenotypes. Strikingly, 15-LOX2, but not 15-LOX2sv-b, transgenic prostate showed upregulation of several well-known stem or progenitor cell molecules including Sca-1, Trop2, p63, Nkx3.1 and Psca. Prostatic hyperplasia caused by both 15-LOX2 and 15-LOX2sv-b did not progress to prostatic intraprostate neoplasia or carcinoma and, mechanistically, prostate lobes (especially those of 15-LOX2 mice) showed a dramatic increase in senescent cells as revealed by increased SA-βgal, p27Kip1 and heterochromatin protein 1γ staining. Collectively, our results suggest that 15-LOX2 expression in mouse prostate leads to hyperplasia and also induces cell senescence, which may, in turn, function as a barrier to tumor development.

In vivo functional studies of tumor-specific retrogene NanogP8 in transgenic animals

The current study was undertaken to investigate potential oncogenic functions of NanogP8, a tumor-specific retrogene homolog of Nanog (expressed in pluripotent cells), in transgenic animal models. To this end, human primary prostate tumor-derived NanogP8 was targeted to the cytokeratin 14 (K14) cellular compartment, and two lines of K14-NanogP8 mice were derived. The line 1 animals, expressing high levels of NanogP8, experienced perinatal lethality and developmental abnormalities in multiple organs, including the skin, tongue, eye, and thymus in surviving animals. On postnatal day 5 transgenic skin, for example, there was increased c-Myc expression and Ki-67+ cells accompanied by profound abnormalities in skin development such as thickened interfollicular epidermis and dermis and lack of hypodermis and sebaceous glands. The line 3 mice, expressing low levels of NanogP8, were grossly normal except cataract development by 4–6 mo of age. Surprisingly, both lines of mice do not develop spontaneous tumors related to transgene expression. Even more unexpectedly, high levels of NanogP8 expression in L1 mice actually inhibited tumor development in a two-stage chemical carcinogenesis model. Mechanistic studies revealed that constitutive NanogP8 overexpression in adult L1 mice reduced CD34+α6+ and Lrig-1+ bulge stem cells, impaired keratinocyte migration, and repressed the expression of many stem cell-associated genes, including Bmp5, Fgfr2, Jmjd1a, and Jun. Our study, for the first time, indicates that transgenically expressed human NanogP8 is biologically functional, but suggests that high levels of NanogP8 may disrupt normal developmental programs and inhibit tumor development by depleting stem cells.

Tumor-suppressive functions of 15-Lipoxygenase-2 and RB1CC1 in prostate cancer

15-Lipoxygenase-2 (15-LOX2) is a human-specific lipid-peroxidizing enzyme most prominently expressed in epithelial cells of normal human prostate but downregulated or completely lost in > 70% of prostate cancer (PCa) cases. Transgenic expression of 15-LOX2 in the mouse prostate surprisingly causes hyperplasia. Here we first provide evidence that 15-LOX2-induced prostatic hyperplasia does not progress to PCa even in p53+/− or p53−/− background. More important, by generating 15-LOX2; Hi-Myc double transgenic (dTg) mice, we show that 15-LOX2 expression inhibits Myc-induced PCa development, such that in the 3-month- and 6-month-old dTg mice, there is a significant reduction in prostate intraneoplasia (PIN) and PCa prevalent in age-matched Hi-Myc prostates. The dTg prostates show increased cell senescence and expression of several senescence-associated molecules, including p27, phosphorylated Rb, and Rb1cc1. We further show that in HPCa, 15-LOX2 and c-Myc manifest reciprocal protein expression patterns. Moreover, RB1CC1 accumulates in senescing normal human prostate (NHP) cells, and in both NHP and RWPE-1 cells, the 15-LOX2 metabolic products 15(S)-HPETE and 15(S)-HETE induce RB1CC1. We finally show that unlike 15-LOX2, RB1CC1 is not lost but rather frequently overexpressed in PCa samples. RB1CC1 knockdown in PC3 cells enhances clonal growth in vitro and tumor growth in vivo. Together, our present studies provide evidence for tumor-suppressive functions for both 15-LOX2 and RB1CC1.

Transgenic overexpression of NanogP8 in the mouse prostate is insufficient to initiate tumorigenesis but weakly promotes tumor development in the Hi-Myc mouse model

This project was undertaken to address a critical cancer biology question: Is overexpression of the pluripotency molecule Nanog sufficient to initiate tumor development in a somatic tissue? Nanog1 is critical for the self-renewal and pluripotency of ES cells, and its retrotransposed homolog, NanogP8 is preferentially expressed in somatic cancer cells. Our work has shown that shRNA-mediated knockdown of NanogP8 in prostate, breast, and colon cancer cells inhibits tumor regeneration whereas inducible overexpression of NanogP8 promotes cancer stem cell phenotypes and properties. To address the key unanswered question whether tissue-specific overexpression of NanogP8 is sufficient to promote tumor development in vivo, we generated a NanogP8 transgenic mouse model, in which the ARR2PB promoter was used to drive NanogP8 cDNA. Surprisingly, the ARR2PB-NanogP8 transgenic mice were viable, developed normally, and did not form spontaneous tumors in >2 years. Also, both wild type and ARR2PB-NanogP8 transgenic mice responded similarly to castration and regeneration and castrated ARR2PB-NanogP8 transgenic mice also did not develop tumors. By crossing the ARR2PB-NanogP8 transgenic mice with ARR2PB-Myc (i.e., Hi-Myc) mice, we found that the double transgenic (i.e., ARR2PB-NanogP8; Hi-Myc) mice showed similar tumor incidence and histology to the Hi-Myc mice. Interestingly, however, we observed white dots in the ventral lobes of the double transgenic prostates, which were characterized as overgrown ductules/buds featured by crowded atypical Nanog-expressing luminal cells. Taken together, our present work demonstrates that transgenic overexpression of NanogP8 in the mouse prostate is insufficient to initiate tumorigenesis but weakly promotes tumor development in the Hi-Myc mouse model.This project was undertaken to address a critical cancer biology question: Is overexpression of the pluripotency molecule Nanog sufficient to initiate tumor development in a somatic tissue? Nanog1 is critical for the self-renewal and pluripotency of ES cells, and its retrotransposed homolog, NanogP8 is preferentially expressed in somatic cancer cells. Our work has shown that shRNA-mediated knockdown of NanogP8 in prostate, breast, and colon cancer cells inhibits tumor regeneration whereas inducible overexpression of NanogP8 promotes cancer stem cell phenotypes and properties. To address the key unanswered question whether tissue-specific overexpression of NanogP8 is sufficient to promote tumor development in vivo, we generated a NanogP8 transgenic mouse model, in which the ARR2PB promoter was used to drive NanogP8 cDNA. Surprisingly, the ARR2PB-NanogP8 transgenic mice were viable, developed normally, and did not form spontaneous tumors in >2 years. Also, both wild type and ARR2PB-NanogP8 transgenic mice responded similarly to castration and regeneration and castrated ARR2PB-NanogP8 transgenic mice also did not develop tumors. By crossing the ARR2PB-NanogP8 transgenic mice with ARR2PB-Myc (i.e., Hi-Myc) mice, we found that the double transgenic (i.e., ARR2PB-NanogP8; Hi-Myc) mice showed similar tumor incidence and histology to the Hi-Myc mice. Interestingly, however, we observed white dots in the ventral lobes of the double transgenic prostates, which were characterized as overgrown ductules/buds featured by crowded atypical Nanog-expressing luminal cells. Taken together, our present work demonstrates that transgenic overexpression of NanogP8 in the mouse prostate is insufficient to initiate tumorigenesis but weakly promotes tumor development in the Hi-Myc mouse model.

Abstract 683: Quantification of rare PD-L1 expressing leukocytes and CTCs in peripheral blood of cancer patients

Background: Expression of PD-L1 on tumor tissue is associated with improved response to PD-1 and PD-L1 checkpoint inhibitors. Additionally, expression of PD-L1 on infiltrating T cells is associated with immune exhaustion. Currently, PD-L1 analysis as well as measures of infiltrating T cells are examined in surgically removed or biopsied samples, usually taken long before clinical decision points. Additionally, given tumor heterogeneity, metastatic lesions are likely to be under-sampled. We sought to examine expression of PD-L1 on circulating tumor cells (CTCs) and leukocytes cell populations with a non-invasive liquid biopsy. Examining dynamic biomarker changes in longitudinal samples could enable the development of novel diagnostic tools for response prediction and pharmacodynamics studies related to immunotherapy. Methods: Blood samples from cancer patients were received and shipped to Epic Sciences. Contrived blood samples were also developed utilizing spike-in of cancer cell line controls into healthy blood samples. RBCs were lysed, and nucleated cells plated onto glass slides utilizing the Epic Sciences process. Slides are stained with an IF cocktail (CK, DAPI, CD45, PD-L1) and scanned. Approximately 3 million nucleated cells are examined through advanced Digital Pathology pipelines to detect rare CTCs (CK+/-, cancer morphology, CD45-) and leukocytes (CK-, CD45+, leukocyte morphology) and analyze for PD-L1 expression. Results: A limit of detection of 1 cell/mL of blood was analytically validated. Using Epic’s rare cell detection platform, we could detect a wide range of PD-L1+ leukocyte counts in a subset of peripheral blood of cancer patients, observed as low as 0.003% (110 of 3,192,263) of total leukocytes. PD-L1+ CTCs were observed in the presence of both high and low counts of PD-L1+ leukocyte populations. Conclusions: Epic Sciences CTC platform’s low limit of detection coupled with ability to archive patient blood samples allowed precise quantification of PD-L1 expression on CTCs and leucocytes retrospectively. In addition, we have previously demonstrated the platform’s ability to detect leucocyte subtypes such as CD3, CD4 and CD8 cells, which will allow us to further characterize PD-L1 expression in T-lymphocytes and other immune cell types. Development of a liquid biopsy based platform that is capable of simultaneously measuring immune biomarkers in CTCs as well as leucocytes will allow real time assessment of response to immune checkpoints inhibitors. Citation Format: Adam Jendrisak, Jiyun Byun, Mahipal Suraneni, David Lu, Rachel Krupa, Sarah Orr, Ryon Graf, Yipeng Wang, Mark Landers, Ryan Dittamore. Quantification of rare PD-L1 expressing leukocytes and CTCs in peripheral blood of cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 683. doi:10.1158/1538-7445.AM2017-683

Abstract 2548: Using whole slide digital image analysis to quantify leukocyte populations in tumor sections

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In recent years, the tumor microenvironment (TME) has been identified as an important factor influencing the growth and metastasis of the tumor. Multiple studies have shown that adding the “Immunoscore” assessment to the AJCC/UICC-TNM classification system improves the accuracy of outcome prediction, and in some cases outperforms traditional TNM staging. In many instances these studies have been performed utilizing 2-3 independent readers to manually quantify the cells, performed on selective high-powered fields or TMA cores rather than the entire specimen, resulting in variations in counts when different high powered fields (HPFs) or cores are chosen. A key method to increase the throughput and to decrease the variability is to utilize whole slide imaging and computerized image analysis to provide leukocyte counts. An image analysis algorithm which can automatically differentiate tumor from stroma would allow rapid quantification of endpoints in each tissue compartment across the whole specimen. To address these issues, Flagship Biosciences has designed proprietary CellMapTM image analysis algorithm tools to develop an Immunoscore-like paradigm for colorectal cancers (CRCs) to potentially provide new and more accurate TME information to aid in interpretation. Utilizing whole slide imaging (WSI) approaches, CellMap™ allows the quantitation of leukocyte populations (e.g., CD3+, CD8+, FoxP3+) automatically across whole tissue sections. Using this algorithm, leukocyte populations were quantified in sections that have been either singly or dually labeled for inflammatory markers. Our preliminary studies indicate that Immunoscore-like scoring paradigm should be established both in tumor areas and in adjacent stroma to provide the most complete information on the biology of the tumor. We compared the use of this approach in tissue microarray (TMA) cores which generally sample areas of dense tumor mass, and compared automated WSI to manual HPF approaches. Accuracy of the algorithm was demonstrated by comparing data from manual counts to algorithm derived counts using high-powered fields. These data support using CellMap™ in the prospective or retrospective assessment of leukocyte subpopulations in whole slides of clinical samples. This approach will diminish variability in counting, expand the types of endpoints determined, and improve the statistical value of these determinations, thereby facilitating robust TME measurements with clinical value. Note: This abstract was not presented at the meeting. Citation Format: Joseph S. Krueger, Brian Laffin, Holger Lange, Anthony Milici, Eric Neeley, Mirza Peljto, Mahipal Suraneni, David Young. Using whole slide digital image analysis to quantify leukocyte populations in tumor sections. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2548. doi:10.1158/1538-7445.AM2014-2548

Abstract 2842: Evaluation of immunohistochemistry assays against c-Met and HGF to guide companion diagnostic decisions

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Establishing reagent specificity during immunohistochemistry (IHC) based biomarker or companion diagnostic (CDx) assays is challenging. Antibody specificity is dictated in part by recognition of 3D confirmation of the target binding, target activity, and and/or epitopes post-translation modifications. Fixation effects pose additional challenges to epitope recognition during IHC assay. For these reasons, different antibodies against the same target biomarker may demonstrate diversity in prevalence, range, and staining patterns over identical specimens. Thus, determining reagent specificity is a critical part of IHC and CDx assay development. Interpretation is further complicated by the pattern of biomarker expression in specific cell types (e.g. tumor v. stroma) or cell compartments (e.g. membrane v. cytosol). These factors may be critical to associate the drugs mechanism of action with efficacy. In the companion diagnostic (CDx) setting, the mechanism of the drug, epitope recognition, and staining features used to interpret and quantify the biomarker to predict patient response requires an evidence-based approach, where all features of an IHC assay are considered and tied empirically to patient response to the drug. In this study, we demonstrate these complexities in gastric cancer specimens, by comparing IHC assays using two antibodies that recognize either intracellular or extracellular domains of c-Met receptor (SP44/ C-term and EP154Y/ N-term) in the context of the ligand for c-Met, Hepatocyte Growth Factor (HGF). Therapeutic antibodies targeting c-Met [such as MetMab® (OA-5D5/ Roche)]; or HGF directly [such as Rilotumumab (AMG 102/ Amgen)], have directly linked c-Met protein expression as revealed by IHC to patient response. Thus, we hypothesized that a link between c-Met protein expression and HGF should be discernible. To test this hypothesis, we used image analysis approaches to determine the staining features of each IHC assay in comparison to each other. Surprisingly, we found little concordance between the two c-Met antibodies in evaluating c-Met and HGF expression. We found distinct populations of c-Met expressing vs HGF expressing specimens, whose HGF-c-Met association differed with the c-Met assay used. We examined the tissue and cell compartmentalization, and identified staining features of each reagent which would aid or impede in interpretation strategies for understanding the relationship between c-Met and HGF. These results suggest that the epitope-specific features of each c-Met antibody determines the relationship with HGF expression, and how quantitative image analysis endpoints can be used to make critical decisions during the development of an IHC companion diagnostic. Note: This abstract was not presented at the meeting. Citation Format: Joseph S. Krueger, Brian Laffin, Holger Lange, Eric Neeley, Mirza Peljto, Mohamed Salama, Mahipal Suraneni, David Young. Evaluation of immunohistochemistry assays against c-Met and HGF to guide companion diagnostic decisions. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2842. doi:10.1158/1538-7445.AM2014-2842

Abstract 4981: Evaluating the contribution of heterogeneity and the tumor microenvironment in companion diagnostic approaches

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA As our understanding of the factors which affect efficacy of a targeted therapy increases, the reliance on histopathological analysis of a biomarker has also increased. This is often due to the necessity to weigh the critical factors of a target or biomarker protein in tissue and cellular context. Currently, histopathologic assessment of tumors which aims to project patient clinical outcome utilize drug target response factors, such as relative expression of the drug target of the drug target or a resistance mechanism in the target (tumor) cells or the tumor microenvironment (TME ). Multiple studies have also shown that TME factors such as inflammatory cell content can be prognostic and predictive. For example, adding the Immunoscore assessment to the TNM classification systems improves the accuracy of disease prognosis. This correlation has led to the concept of predictive “immunoprofiling”, which uses an individuals immune system profile to predict that patients response to immunomodulating antibody therapy. For these reasons, it is critical to evaluate the drug target or biomarker in conjunction with TME features when defining a patient selection strategy. Critical factors such as tissue and/or cellular compartmentalization and tumor heterogeneity direct the interpretation of these measures and their predictive value. To address the need for careful, contextual interpretation of histopathological evaluations, Flagship has built CellMap™ image analysis algorithms to directly measure heterogeneity and the TME components in a whole tissue section. These approaches allow biomarker interpretation in the complex context of spatial, architectural, and morphological information to aid histological definition and quantification. In this study, we utilized a cohort of specimens from 20 colorectal cancer (CRC) patients and immunologically stained them in order to visualize c-Met, as a characteristic and biologically relevant therapy target; and CD3+ and CD8+ to visualize the inflammatory cell environment. Using these immunohistochemical markers as a prototype for a simultaneous evaluation of a molecular target and the TME, we characterized the biomarker and inflammatory content in both the tumor and stroma using our CellMap™ image analysis algorithms. This provided detailed accounting of the molecular target profile (tumor vs stroma; membrane vs cytoplasm vs nucleus), and the “immunoprofile”, which allowed us to quantitatively describe and associate these features relative to each other in the context of heterogeneity. This data demonstrated discrete patterns of association between c-met and the TME, serving as a potentially critical measure which reflects a biological process relevant to disease outcome. These studies demonstrate novel tools which can assess both the prognostic and predictive value of key measurements which reflect complex tumor biology. Citation Format: Joseph S. Krueger, Brian Laffin, Holger Lange, Anthony Milici, Mirza Peljto, Eric Neeley, Mahipal Suraneni, David Young. Evaluating the contribution of heterogeneity and the tumor microenvironment in companion diagnostic approaches. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4981. doi:10.1158/1538-7445.AM2014-4981