Andrea E. Knowlton

Spatial Distribution of Blood Vessels and CD34+ Hematopoietic Stem and Progenitor Cells Within the Marrow Cavities of Human Cancellous Bone

Current bone marrow dosimetry methods inherently assume that the target cells of interest for the assessment of leukemia risk (stochastic effects) or marrow toxicity (deterministic effects) are uniformly localized throughout the marrow cavities of cancellous bone. Previous studies on mouse femur, however, have demonstrated a spatial gradient for the hematopoietic stem and progenitor cells, with higher concentrations near the bone surfaces. The objective of the present study was to directly measure the spatial concentration of these cells, as well as marrow vasculature structures, within images of human disease-free bone marrow. Methods: Core-biopsy samples of normal bone marrow from the iliac crest were obtained from clinical cases at Shands Hospital at the University of Florida Department of Pathology. The specimens were sectioned and immunohistochemically stained for CD34 (red) and CD31 (brown) antigens. These 2 stains were used simultaneously to differentiate between hematopoietic stem and progenitor cells (CD34+/CD31−) and vascular endothelium (CD34+/CD31+). Distances from hematopoietic CD34+ cells and blood vessels to the nearest bone trabecula surface were measured digitally and then binned in 50-μm increments, with the results then normalized per unit area of marrow tissue. The distances separating hematopoietic CD34+ cells from vessels were also tallied. Results: Hematopoietic CD34+ cells were found to exist along a linear spatial gradient with a maximal areal concentration localized within the first 50 μm of the bone surfaces. An exponential spatial concentration gradient was found in the concentration of blood vessel fragments within the images. Distances between hematopoietic CD34+ cells and blood vessels exhibited a lognormal distribution indicating a shared spatial niche. Conclusion: Study results confirm that the spatial gradient of hematopoietic stem and progenitor cells previously measured in mouse femur is also present within human cancellous bone. The dosimetric implication of these results may be significant for those scenarios in which the absorbed dose itself is nonuniformly delivered across the marrow tissues, as would be the case for a low-energy β- or α-particle emitter localized on the bone surfaces.

Chlamydial infection induces host cytokinesis failure at abscission.

Chlamydia trachomatis is an obligate intracellular bacteria and the infectious agent responsible for the sexually transmitted disease Chlamydia. Infection with Chlamydia can lead to serious health sequelae such as pelvic inflammatory disease and reproductive tract scarring contributing to infertility and ectopic pregnancies. Additionally, chlamydial infections have been epidemiologically linked to cervical cancer in patients with a prior human papilomavirus (HPV) infection. Chlamydial infection of cultured cells causes multinucleation, a potential pathway for chromosomal instability. Two mechanisms that are known to initiate multinucleation are cell fusion and cytokinesis failure. This study demonstrates that multinucleation of the host cell by Chlamydia is entirely due to cytokinesis failure. Moreover, cytokinesis failure is due in part to the chlamydial effector CPAF acting as an anaphase promoting complex mimic causing cells to exit mitosis with unaligned and unattached chromosomes. These lagging and missegregated chromosomes inhibit cytokinesis by blocking abscission, the final stage of cytokinesis.

Multinucleation during C. trachomatis infections is caused by the contribution of two effector pathways.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen and the second leading cause of sexually transmitted infections in the US. Infections cause significant morbidity and can lead to serious reproductive sequelae, including an epidemiological link to increased rates of reproductive cancers. One of the overt changes that infected cells exhibit is the development of genomic instability leading to multinucleation. Here we demonstrate that the induction of multinucleation is not conserved equally across chlamydial species; C. trachomatis L2 caused high levels of multinucleation, C. muridarum intermediate levels, and C. caviae had very modest effects on multinucleation. Our data show that at least two effector pathways together cause genomic instability during infection leading to multinucleation. We find that the highly conserved chlamydial protease CPAF is a key effector for one of these pathways. CPAF secretion is required for the loss of centrosome duplication regulation as well as inducing early mitotic exit. The second effector pathway involves the induction of centrosome position errors. This function is not conserved in three chlamydial species tested. Together these two pathways contribute to the induction of high levels of genomic instability and multinucleation seen in C. trachomatis infections.

Chlamydia induces anchorage independence in 3T3 cells and detrimental cytological defects in an infection model.

Chlamydia are Gram negative, obligate intracellular bacterial organisms with different species causing a multitude of infections in both humans and animals. Chlamydia trachomatis is the causative agent of the sexually transmitted infection (STI) Chlamydia, the most commonly acquired bacterial STI in the United States. Chlamydial infections have also been epidemiologically linked to cervical cancer in women co-infected with the human papillomavirus (HPV). We have previously shown chlamydial infection results in centrosome amplification and multipolar spindle formation leading to chromosomal instability. Many studies indicate that centrosome abnormalities, spindle defects, and chromosome segregation errors can lead to cell transformation. We hypothesize that the presence of these defects within infected dividing cells identifies a possible mechanism for Chlamydia as a cofactor in cervical cancer formation. Here we demonstrate that infection with Chlamydia trachomatis is able to transform 3T3 cells in soft agar resulting in anchorage independence and increased colony formation. Additionally, we show for the first time Chlamydia infects actively replicating cells in vivo. Infection of mice with Chlamydia results in significantly increased cell proliferation within the cervix, and in evidence of cervical dysplasia. Confocal examination of these infected tissues also revealed elements of chlamydial induced chromosome instability. These results contribute to a growing body of data implicating a role for Chlamydia in cervical cancer development and suggest a possible molecular mechanism for this effect.

Nicotine Reduces Survival via Augmentation of Paracrine HGF–MET Signaling in the Pancreatic Cancer Microenvironment

Purpose: The relationship between smoking and pancreatic cancer biology, particularly in the context of the heterogeneous microenvironment, remains incompletely defined. We hypothesized that nicotine exposure would lead to the augmentation of paracrine growth factor signaling between tumor-associated stroma (TAS) and pancreatic cancer cells, ultimately resulting in accelerated tumor growth and metastasis. Experimental Design: The effect of tobacco use on overall survival was analyzed using a prospectively maintained database of surgically resected patients with pancreatic cancer. Nicotine exposure was evaluated in vitro using primary patient–derived TAS and pancreatic cancer cells independently and in coculture. Nicotine administration was then assessed in vivo using a patient-derived pancreatic cancer xenograft model. Results: Continued smoking was associated with reduced overall survival after surgical resection. In culture, nicotine-stimulated hepatocyte growth factor (HGF) secretion in primary patient-derived TAS and nicotine stimulation was required for persistent pancreatic cancer cell c-Met activation in a coculture model. c-Met activation in this manner led to the induction of inhibitor of differentiation-1 (Id1) in pancreatic cancer cells, previously established as a mediator of growth, invasion and chemoresistance. HGF-induced Id1 expression was abrogated by both epigenetic and pharmacologic c-Met inhibition. In patient-derived pancreatic cancer xenografts, nicotine treatment augmented tumor growth and metastasis; tumor lysates from nicotine-treated mice demonstrated elevated HGF expression by qRT-PCR and phospho-Met levels by ELISA. Similarly, elevated levels of phospho-Met in surgically resected pancreatic cancer specimens correlated with reduced overall survival. Conclusions: Taken together, these data demonstrate a novel, microenvironment-dependent paracrine signaling mechanism by which nicotine exposure promotes the growth and metastasis of pancreatic cancer. Clin Cancer Res; 22(7); 1787–99. ©2015 AACR.

Chlamydia trachomatis homotypic inclusion fusion is promoted by host microtubule trafficking

BackgroundThe developmental cycle of the obligate intracellular pathogen Chlamydia is dependant on the formation of a unique intracellular niche termed the chlamydial inclusion. The inclusion is a membrane bound vacuole derived from host cytoplasmic membrane and is modified significantly by the insertion of chlamydial proteins. A unique property of the inclusion is its propensity for homotypic fusion. The vast majority of cells infected with multiple chlamydial elementary bodies (EBs) contain only a single mature inclusion. The chlamydial protein IncA is required for fusion, however the host process involved are uncharacterized.ResultsHere, through live imaging studies, we determined that the nascent inclusions clustered tightly at the cell microtubule organizing center (MTOC) where they eventually fused to form a single inclusion. We established that factors involved in trafficking were required for efficient fusion as both disruption of the microtubule network and inhibition of microtubule trafficking reduced the efficiency of fusion. Additionally, fusion occurred at multiple sites in the cell and was delayed when the microtubule minus ends were either no longer anchored at a single MTOC or when a cell possessed multiple MTOCs.ConclusionsThe data presented demonstrates that efficient homotypic fusion requires the inclusions to be in close proximity and that this proximity is dependent on chlamydial microtubule trafficking to the minus ends of microtubules.

Isolation of Pancreatic Cancer Cells from a Patient-Derived Xenograft Model Allows for Practical Expansion and Preserved Heterogeneity in Culture

Commercially available, highly passaged pancreatic cancer (PC) cell lines are of limited translational value. Attempts to overcome this limitation have primarily consisted of cancer cell isolation and culture directly from human PC specimens. However, these techniques are associated with exceedingly low success rates. Here, we demonstrate a highly reproducible culture of primary PC cell lines (PPCLs) from patient-derived xenografts, which preserve, in part, the intratumoral heterogeneity known to exist in PC. PPCL expansion from patient-derived xenografts was successful in 100% of attempts (5 of 5). Phenotypic analysis was evaluated with flow cytometry, immunofluorescence microscopy, and short tandem repeat profiling. Importantly, tumorigenicity of PPCLs expanded from patient-derived xenografts was assessed by subcutaneous injection into nonobese diabeteic.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ mice. Morphologically, subcutaneous injection of all PPCLs into mice yielded tumors with similar characteristics to the parent xenograft. PPCLs uniformly expressed class I human leukocyte antigen, epithelial cell adhesion molecule, and cytokeratin-19. Heterogeneity within each PPCL persisted in culture for the frequency of cells expressing the cancer stem cell markers CD44, CD133, and c-Met and the immunologic markers human leukocyte antigen class II and programmed death ligand 1. This work therefore presents a reliable method for the rapid expansion of primary human PC cells and, thereby, provides a platform for translational investigation and, importantly, potential personalized therapeutic approaches.

Abstract 1017: Pro-inflammatory cytokine secretion and gene networks associate with pancreatic cancer induced cachexia

Introduction: Pancreatic cancer (PC) is associated with a high rate of cachexia, which specifically diminishes quality of life, prohibits effective therapies, and subsequently contributes to morbidity and mortality. Unfortunately, mechanisms underlying this cancer-induced muscle wasting in the human disease remain incompletely described, in part due to limited translational models. Therefore, we hypothesize that the development of more representative models of PC cachexia will allow for development of therapeutic targets for cachexia. To test our hypothesis, we propose to 1) establish the first patient-derived xenograft (PDX) cancer cachexia models to identify the muscle associated with PC induced cachexia and 2) support these results by examining the corresponding skeletal muscle of PC patients whom contributed to the PDX models. Methods: Rectus abdominis muscle was biopsied from surgically resected PC patients and matched non-cancer controls. After surgical harvest of PC specimen, PDX models were derived in NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice and skeletal muscle was subsequently harvested for histologic investigations on ultrastructural disorganization and qRT-PCR for atrophy-related transcription factors differentially regulated in PC patients. Systemic cytokine expression profiles were analyzed with luminex technology and confirmed by ELISA. Results: Rectus biopsies from patients with resected PC displayed marked muscle fiber atrophy, increased extracellular space, greater variation in fiber size and shape and more centralized nuclei compared to controls. These architectural abnormalities were also present in mice bearing xenografts from corresponding PC patients. Despite the absence of an adaptive immune system, PDX mice demonstrated high levels of systemic TNFα, IL-1β, IL6 and KC (IL8) with a concomitant decrease in anti-inflammatory cytokine IL10 when compared to matched controls. Further, skeletal muscle from both patients with PC and mice bearing PDX tumors demonstrated increased expression of the Forkhead boxO1 (FoxO1) transcription factor and FoxO target gene and E3 ubiquitin ligase, MuRF1, both of which have been directly implicated in the regulation of muscle mass. Conclusions: Preoperative muscle wasting in PC is associated with characteristic architectural abnormalities and elevated FoxO1-MuRF1 levels. Mice bearing PDX demonstrate comparable elevations in circulating pro-inflammatory cytokines, muscle pathology and FoxO1-MuRF1 levels. These results provide a valid translational model of cachexia which suggests a central role for FoxO1 and MuRF1 in PC-associated muscle wasting. Citation Format: Daniel Delitto, Sarah M. Judge, Rachel L. Nosacka, Andrea Knowlton, Fernanda G. Rocha, Kevin E. Behrns, Steven J. Hughes, Shannon M. Wallet, Andrew R. Judge, Jose G. Trevino. Pro-inflammatory cytokine secretion and gene networks associate with pancreatic cancer induced cachexia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1017.

Abstract 470: Nicotine reduces survival via augmentation of paracrine HGF-MET signaling in the pancreatic cancer microenvironment

Smoking is an established risk factor for the development of pancreatic adenocarcinoma (PC). However, the relationship between smoking and PC tumor biology is incompletely defined. We report reduced overall survival (OS) in PC patients who continued smoking after surgical resection with curative intention (HR 1.93; P = .040). We further demonstrate augmented paracrine signaling via the hepatocyte growth factor (HGF)/c-Met pathway as a result of nicotine exposure in the PC microenvironment. Specifically, HGF, secreted by patient-derived PC tumor associated stroma (TAS), activates the c-Met receptor in PC cells. This paracrine activation subsequently leads to downstream induction of inhibitor of differentiation-1 (Id1) in PC cells, previously established as a mediator of chemoresistance. Further delineation of the signaling pathway demonstrates HGF-induced Id1 expression is abrogated by silencing of c-Met or pharmacologic inhibition. In patient-derived PC xenografts, nicotine treatment augmented tumor growth and metastasis; tumor lysates from nicotine-treated mice demonstrated elevated HGF expression and phospho-Met levels. Additionally, patients with high intratumoral phospho-Met levels exhibited reduced overall survival compared to those with low phospho-Met levels (Median OS 6.1 vs. 15.2 months, respectively; P = .028). Taken together, our data reveal that nicotine promotes the progression of PC via a microenvironment-dependent, paracrine signaling mechanism. Citation Format: Daniel Delitto, Dongyu Zhang, Song Han, Brian S. Black, Andrea E. Knowlton, Adrian C. Vlada, George A. Sarosi, Kevin E. Behrns, Ryan M. Thomas, Xiaomin Lu, Chen Liu, Thomas J. George, Steven J. Hughes, Shannon M. Wallet, Jose G. Trevino. Nicotine reduces survival via augmentation of paracrine HGF-MET signaling in the pancreatic cancer microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 470. doi:10.1158/1538-7445.AM2015-470