Supplemental Figure S2. Nicotine-mediated induction of c-Met is Src-dependent.

Nicotine treatment does not lead to c-Met activation in PC cells.

Supplemental Figure S1. Induction of c-Met by nicotine occurs at the post-transcriptional level.

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 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-PrkdcscidIl2rgtm1Wjl/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. Treviño. 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.

The cancer microenvironment allows tumor cells to evade immune surveillance through a variety of mechanisms. While interferon-γ (IFNγ) is central to effective antitumor immunity, its effects on the microenvironment are not as clear and have in some cancers been shown to induce immune checkpoint ligands. The heterogeneity of these responses to IFNγ remains poorly characterized in desmoplastic malignancies with minimal inflammatory cell infiltration, such as pancreatic cancer (PC). Thus, the IFNγ response within and on key cells of the PC microenvironment was evaluated. IFNγ induced expression of human leukocyte antigen (HLA) class I and II on PC cell lines, primary pancreatic cancer epithelial cells (PPCE) and patient-derived tumor-associated stroma, concomitant with an upregulation of PDL1 in the absence of CD80 and CD86 expression. As expected, IFNγ also induced high levels of CXCL10 from all cell types. In addition, significantly higher levels of CXCL10 were observed in PC specimens compared to those from chronic pancreatitis, whereby intratumoral CXCL10 concentration was an independent predictor of poor survival. Immunohistochemical analysis revealed a subset of CXCR3-positive cancer cells in over 90 % of PC specimens, as well as on a subset of cultured PC cell lines and PPCE, whereby exposure to CXCL10 induced resistance to the chemotherapeutic gemcitabine. These findings suggest that IFNγ has multiple effects on many cell types within the PC microenvironment that may lead to immune evasion, chemoresistance and shortened survival.

Abstract 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.

Abstract The systemic treatment of pancreatic cancer (PC) is hindered by the rapid development of chemoresistance to current cytotoxic therapies. Mechanisms governing the development of chemoresistance remain poorly characterized, particularly with respect to contributions from the tumor microenvironment. Here, intratumoral soluble mediator concentrations from resected PC specimens (n=26) as well as supernatants from co-cultures of PC cell lines and primary tumor-associated pancreatic stellate cells (PSCs) (n=16) were evaluated using a panel of 43 growth factors, chemokines and cytokines. CXCL10 levels were significantly increased in PC specimens as well as upon co-culture of PC cells with tumor-associated PSCs. In addition, high intratumoral CXCL10 concentrations correlated with reduced overall survival (HR 6.9; P = .006). Thus, the effect of CXCL10 on viability, proliferation, and apoptosis of PC cell lines was evaluated with and without gemcitabine treatment. While CXCL10 treatment had a small effect on the viability of PC cell lines, it significantly increased viability of these cells in the presence of gemcitabine. In addition, gemcitabine treatment induced the expression of the CXCL10 receptor, CXCR3. Thus, paracrine CXCL10 signaling between stromal, PC and immune cells within the pancreatic cancer microenvironment the may be responsible for chemoresistance to gemcitabine.

Abstract Background: The systemic treatment of pancreatic cancer (PC) is hindered by the rapid development of chemoresistance to current cytotoxic therapies. Mechanisms governing the development of chemoresistance remain poorly characterized, particularly with respect to contributions from the tumor microenvironment. Thus, the goal of this study was to identify novel mechanisms acting within the tumor microenvironment which lead to PC chemoresistance. Methods: Intratumoral soluble mediator concentrations from resected PC specimens (n = 26) as well as supernatants from co-cultures of primary tumor-associated pancreatic stellate cells (PSCs) and PC cells (n = 12) were evaluated using a panel of 41 growth factors, chemokines and cytokines. The effect of CXCL10, a highly expressed soluble mediator during co-culture, on viability, proliferation, and apoptosis of PC cells was evaluated with and without gemcitabine treatment. In addition, the contribution of CXCL10 on migration patterns of peripheral blood mononuclear cells (PBMCs) was assessed. Results: Co-culture of tumor-associated PSCs with PC cells revealed increased CXCL10 levels compared to either cell type cultured alone. In addition, high intratumoral CXCL10 concentrations correlated with reduced overall survival (HR 6.9; P = .006). While CXCL10 treatment had a small effect on the viability of PC cells, it led to significantly increased PC cell viability in the presence of gemcitabine. Further, gemcitabine treatment induced the expression of the CXCL10 receptor, CXCR3, and this induction of CXCR3 was associated with the absence of apoptotic markers in PC cells. Finally, constitutive expression of CXCL10 by PC cells preferentially led to the migration of regulatory immune cell subsets. Conclusion: Paracrine CXCL10 signaling between stromal, PC and immune cells may be responsible not only for chemoresistance to gemcitabine, but also the recruitment and potential polarization of regulatory immune cell subsets in the pancreatic cancer microenvironment. Citation Format: Daniel Delitto, Chelsey Perez, Brian S. Black, Heather L. Sorenson, Andrea E. Knowlton, Song Han, Dongyu Zhang, George A. Sarosi, Lyle L. Moldawer, Kevin E. Behrns, Chen Liu, Thomas J. George, Ryan M. Thomas, Jose G. Trevino, Shannon M. Wallet, Steven J. Hughes. CXCL10 within the tumor microenvironment induces gemcitabine resistance in pancreatic cancer cells. [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 5028. doi:10.1158/1538-7445.AM2015-5028

The tumor microenvironment impacts pancreatic cancer (PC) development, progression and metastasis. How intratumoral inflammatory mediators modulate this biology remains poorly understood. We hypothesized that the inflammatory milieu within the PC microenvironment would correlate with clinicopathologic findings and survival.

Pancreatic cancer is the fourth leading cause of cancer deaths in the United States and one of the most difficult-to-treat cancers. Despite advances in chemotherapy, 5-year survival following surgical resection is only 23% in specialized pancreatic surgery centers. A major stumbling block for these and other cancer treatments is a way to efficiently deliver therapeutics to the target cells. Thus, we have identified high affinity molecular targeting molecules unique to an individual pancreatic cancer, which were generated utilizing a high throughput system using fresh surgical specimens along with phage display to develop novel small chain variable fragments (scFv). Here we present the characterization of two scFv molecules identified, through which we demonstrate by ELISA and BioLayer Interferometry that our scFv bind with high specificity to the neoplasm from which they were derived, while remaining minimally reactive to healthy pancreatic tissue. In addition, we have engineered these scFV to be assembled into a homotrimeric alpha-helical coiled coil using a novel platform in order to retain its multimeric nature. Future studies will assess the ability of these scFv to home to the original neoplasm using our established patient-derived tumor xenograft models whereby the human tumor is grown in NOD/SCID mice. Together these data will determine whether screening individual neoplasms using phage display can be used to develop scFv as personalized targeting molecules.

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 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.

BACKGROUND:The 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.RESULTS:Here, 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.CONCLUSIONS:The 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.

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.

Chlamydiae are Gram negative, obligate intracellular bacteria, and Chlamydia trachomatis is the etiologic agent of the most commonly reported sexually transmitted disease in the United States. Chlamydiae undergo a biphasic life cycle that takes place inside a parasitophorous vacuole termed an inclusion. Chlamydial infections have been epidemiologically linked to cervical cancer in patients previously infected by human papillomavirus (HPV). The inclusion associates very closely with host cell centrosomes, and this association is dependent upon the host motor protein dynein. We have previously reported that this interaction induces supernumerary centrosomes in infected cells, leading to multipolar mitotic spindles and inhibiting accurate chromosome segregation. Our findings demonstrate that chlamydial infection causes mitotic spindle defects independently of its effects on centrosome amplification. We show that chlamydial infection increases centrosome spread and inhibits the spindle assembly checkpoint delay to disrupt centrosome clustering. These data suggest that chlamydial infection exacerbates the consequences of centrosome amplification by inhibiting the cells' ability to suppress the effects of these defects on mitotic spindle organization. We hypothesize that these combined effects on mitotic spindle architecture identifies a possible mechanism for Chlamydia as a cofactor in cervical cancer formation.

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.