Orthotopic Sites Research Articles

Abstract A140: Check point inhibitor modulation of tumor microenvironment at orthotopic and metastatic sites using bioluminescent syngeneic cell line models in immune competent mice

Abstract Introduction: Syngeneic models are widely used to model the impact of cancer immunotherapy on tumor growth and tumor invading leucocytes (TILs), and the majority of such work is typically carried out in the subcutaneous setting. However, orthotopic models are known to better model cancer in patients as they form a single focal disease area as in the patient situation, facilitate metastatic spread via intra- and extra-thoracic lymph nodes and, in the case of syngeneic models, strain-specific tumor microenvironment interactions (immune and stromal components). Bioluminescent imaging (BLI) increases the usefulness of such models, as it allows for non-invasive longitudinal monitoring of tumor burden, allowing for optimal randomisation and reduction of false positives. It also allows continuous feedback allowing one to optimise treatment regimen mid-study. Herein we describe the generation of several bioluminescent variants of syngeneic cell lines commonly used for immunotherapy studies and assess the impact of orthotopic growth on response to immune check point therapy. Experimental Procedures: Bioluminescent cell line variants of syngeneic cell lines were established by lentiviral transduction for: 4T1 (breast), B16-F10 (melanoma), MBT-2 (bladder) H22 (Hepatoma), LL/2 (lung), Pan02 (Pancreatic) and RM-1 (prostate). Following establishment of stable cultures, DNA profiling and in vitro cytotoxicity assays was carried out to ensure there was no significant changes in DNA, cell doubling time or response to SoC agents following transduction. Subcutaneous growth of wild-type and bioluminescent variants was compared to assess any impact of luciferase expression on tumor growth, inflammation and TILs. Orthotopic models were established for most cell lines, and a metastatic model for B16-F10, BLI was carried out to assess real-time tumor growth and tumor burden at end stage (Spectrum CT; PerkinElmer). Immune checkpoint therapy was also assessed and TIL infiltration by FACs analysis and IHC. Results: Stable transduced bioluminescent cell lines were established; cell doubling time, morphology and growth in vitro was found to be consistent with their wild-type counterparts. Bioluminescent 4T1 cells exhibited growth consistent to that previously reported and readily metastasised to the lungs from both the orthotopic and subcutaneous sites; B16-F10 cells readily metastasise to bone following intracardiac administration. Subcutaneous response to immunotherapy did not appear to be affected. Modulation of immune cell infiltration will be reported and correlated to response. Conclusions: The growth and response to immunotherapy does not appear to be significantly impacted in the bioluminescent cell line models tested; as such they are a useful tool for further assessing the impact of complex orthotopic, spontaneous and experimental metastasis modelling in immune competent mice. Citation Format: Andrew McKenzie, Nektaria Papadopoulou, Simon Jiang, Jane Wrigley, Kelly Jones, Russell Garland, Neil Williams, Rajendra Kumari. Check point inhibitor modulation of tumor microenvironment at orthotopic and metastatic sites using bioluminescent syngeneic cell line models in immune competent mice [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A140.

Comparison of the Adipose and Luminal Mammary Gland Compartment as Orthotopic Inoculation Sites in a 4T1-Based Immunocompetent Preclinical Model for Triple-Negative Breast Cancer.

Breast tumorigenesis is classically studied in mice by inoculating tumor cells in the fat pad, the adipose compartment of the mammary gland. Alternatively, the mammary ducts, which constitute the luminal mammary gland compartment, also provide a suitable inoculation site to induce breast cancer in murine models. The microenvironments in these compartments influence tumor cell progression, yet this effect has not been investigated in an immunocompetent context. Here, we compared both mammary gland compartments as distinct inoculation sites, taking into account the immunological aspect by inoculating 4T1 tumor cells in immunocompetent mice. Following tumor cell inoculation in the adipose compartment of non-pretreated/naive, hormonally pretreated/naive and non-pretreated/lactating mice, the primary tumors developed similarly. However, a slower onset of primary tumor growth was found after inoculations in the luminal compartment of non-pretreated/lactating mice. Despite this difference in tumor development rate, metastasis to the liver and lungs was equally observed and was accompanied by lymphatic spreading of tumor cells and progressive splenomegaly with both inoculation types. Chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) served as innovative biomarkers for disease progression showing increased levels in primary tumors and sera of the non-pretreated/lactating inoculation groups. A slower increase in circulating CHI3L1 but not LCN2 levels, was observed after inoculations in the luminal compartment which corroborated the slower tumor development at this inoculation site. Our results highlight the critical impact of different mammary gland compartments on tumor development in syngeneic murine models and support the use of novel tumor progression biomarkers in an immune-competent environment.

Real-Time GFP Intravital Imaging of the Differences in Cellular and Angiogenic Behavior of Subcutaneous and Orthotopic Nude-Mouse Models of Human PC-3 Prostate Cancer.

There are two major types of mouse xenograft models of cancer: subcutaneous implantation and orthotopic implantation. Subcutaneous transplant models are widely used with both cancer cell lines and human-tumor specimens. Recently, subcutaneous models of patient tumors, termed patient-derived xenographs (PDX) have become highly popular and have acquired such names as "Avatar" and "Xenopatients." However, such s.c. models rarely metastasize and are therefore not patient-like. In contrast, orthotopic models have the capability to metastasize. If intact fragments of tumor tissue are implanted by surgical orthotopic implantation (SOI), the metastatic potential can match that of the donor patient. The present study images in real time, using green fluorescent protein (GFP) expression, the very different tumor behavior at the orthotopic and subcutaneous sites of human prostate cancer PC-3 in athymic nude mice. By day-2 after tumor implantation, the orthotopic tumor is already highly vascularized and the cancer cells have begun to migrate out of the tumor. In contrast, the subcutaneous tumor only begins to be vascularized by day-3 and cells do not migrate from the tumor. Angiogenesis is much more extensive in the orthotopic tumor throughout the 2-week observation period. The orthotopic PC-3-GFP tumor progresses very rapidly and distinct metastasis have appeared in lymph nodes by day-3 which rapidly appear in many areas of the abdominal cavity including portal lymph nodes by day-7. At day-14, no invasion or metastasis was observed with the s.c. tumor even when the animal was extensively explored. These results explain why orthotopic tumors mimimc clinical metastatic tumors in nude mice and why subcutaneous tumors do not. J. Cell. Biochem. 117: 2546-2551, 2016. © 2016 Wiley Periodicals, Inc.

Abstract B31: Establishment and molecular characterization of patient-derived tumor xenografts from resected tumors or ascites fluids of patients with pancreatic/ampullary/bile duct carcinomas

Abstract Pancreatic adenocarcinoma (PDAC) is the 4th most common cause of cancer deaths in North America, for both men and women with a 5-year survival rate of less than 5%. The poor prognosis rate is attributed to late presentation of the disease and the lack of effective treatment options. Large-scale genome sequencing efforts on PDAC tumors show evidence of high mutational burden and revealed a number of mutated genes affecting multiple oncogenic pathways. While there are significant endeavors in developing specific targeted agents against “driver” mutations, tumor diversity within and across patient population remains a key factor affecting therapeutic efficacy. In this context, the availability of large cohorts of genomically characterized patient-derived xenograft (PDX) tumor models may help to accelerate the development of novel therapies against this lethal cancer. PDX models provide a renewable resource to maintain a patient's tumor ex vivo for pre-clinical or co-clinical studies. As part of The International Cancer Genome Consortium (ICGC), our laboratory has established 93 PDX models in non-obese diabetic and severe combined immune-deficient (NOD-SCID) mice from Whipple resection specimens. These tumors represent a heterogeneous group of neoplasms arising from the head, body and tail of pancreas, bile duct and Ampulla of Vater. All implantations including in the subcutaneous pocket at the flank or at the orthotopic pancreas site, were performed using 4-8 weeks old NOD-SCID mice. Successful growth and serial transplant to multiple mouse generations were observed in in 74 PDX models of the 93 implanted PDAC specimens, achieving an 80% engraftment rate, one of the highest reported in any type of cancer. Histology fidelity was preserved in the PDX models compared to corresponding patient tumors. Failed implants were due to specimens characterized by borderline malignancy and absence of tumor cells. Whole exome sequencing and copy number aberration profiling was completed for 61 PDXs and blood from the matched patients. Cancer-specific single nucleotide variation (SNV) load varied widely from 38 to 305 in PDXs. The most recurrent activating mutation was observed in KRAS with 77% of PDX models showing alterations at codon G12 (65%), G13 (8%) and Q61 (4%); in addition, 26% PDXs had a copy number gain in KRAS. Molecular comparisons of the 21 PDX models and their matched patient tumors showed that alternate allele frequency of KRAS mutation from exome sequencing of primary tumor is a strong indicator of the tumor cellularity; a higher tumor cellularity results in a larger overlap of cancer specific alterations between xenografts and corresponding patient tumors. We have demonstrated a successful establishment of PDX models that represent genomic architecture of major subclonal populations of patient PDAC primary tumors. Citation Format: Nikolina Radulovich, Emin Ibrahimov, Carson Holt, Vibha Raghavan, Tracy Zhao, Rob Denroch, Nhu-An Pham, Steve Gallinger, Melania Pintilie, Lincoln Stein, John McPherson, Lakshmi Muthuswamy, Ming Sound Tsao. Establishment and molecular characterization of patient-derived tumor xenografts from resected tumors or ascites fluids of patients with pancreatic/ampullary/bile duct carcinomas. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B31.

Abstract 2907: Eugenol potentiates the effect of cisplatin on cancer stem-like cells through targeting the NF-κB pathway

Abstract Triple-negative subtype of breast cancer (TNBC) contains exclusively tumorigenic and often endowed with self-renewing and resistance to cytotoxic chemotherapy, such as, paclitaxel resulting in relative increases in CSCs phenotype. Signaling through NF-kB may be essential for the CSCs self-renewal and could present potential target for novel treatment in breast cancer patients. We explored the possible involvement of NF-kB pathway that contribute to the functions and maintenance of breast CSCs and targeting this pathway by cisplatin combining with a natural product eugenol both in vitro and in vivo, using estrogen dependent (MCF-7) and independent (MDA-MB-231, MDA-MB-468 and BT-20) cell lines and xenografted both subcutaneous and orthotopic (fat pad) sites in nude mice. Chromatin immunoprecipitation (ChIP) assay was utilized to assess the NF-kB associated DNA binding capacity in promoter region. Surprisingly, neither cisplatin nor eugenol was capable of complete diminishing the CSCs populations. Cisplatin alone administration partially diminished apoptosis, mammospheres formation, aldehyde dehydrogenase 1 (ALDH1) activity, invasion and growth on nude mice. We observed cisplatin-induced activation of NF-kB associated with survival and regrowth of mamospheres. For effective elimination of CSCs phenotype by cisplatin, we cotreated cells with eugenol. Combination of eugenol and cisplatin significantly eradicated cisplatin induced NF-kB DNA binding capability assed by EMSA and ChIP assays, which was associated with abrogated apoptosis, mammospheres formation, ALDH1 activity and invasion. In vivo, combination therapy reduced the tumor size in synergistic manner. This was possibly due to induction of apoptosis, inhibition of proliferation, angiogenesis and downregulation of cisplatin-induced expression of proteins of NF-kB target genes. Although treatment of mammospheres by cisplatin partially induced the CSCs population through interleukin (IL)-6 pathway, however, only coadministration of eugenol and cisplatin reduced the numbers of CSCs to virtually undeletable levels in vitro and in vivo. Our data suggest that eugenol may be well suited to increase targeting of breast CSCs by cisplatin. Citation Format: Syed S. Islam, Al-Sharif Ibtehaj, Sultan Ahlam, Abdelilah Aboussekhra. Eugenol potentiates the effect of cisplatin on cancer stem-like cells through targeting the NF-κB pathway. [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 2907.

Human Induced Pluripotent Stem Cells Differentiate Into Functional Mesenchymal Stem Cells and Repair Bone Defects.

Mesenchymal stem cells (MSCs) are currently the most established cells for skeletal tissue engineering and regeneration of various skeletal conditions; however, availability of autologous MSCs is very limited. This study demonstrates a new method to differentiate human fibroblast-derived induced pluripotent stem cells (iPSCs) to cells with MSC properties, which we comprehensively characterized including differentiation potential and transcriptomic analysis. We showed that these iPS-derived MSCs are able to regenerate nonunion bone defects in mice more efficiently than bone marrow-derived human MSCs when overexpressing BMP6 using a nonviral transfection method.

Abstract A08: A patient-derived xenograft model to study the effects of postmenopausal obesity on endocrine therapy resistance

Abstract Introduction: Obesity affects more than one-third of adults in the U.S. and increases the risk for and worsens outcomes of individuals with cancer. Obese women have a greater risk of postmenopausal hormone-receptor positive breast cancer and are more likely to relapse after endocrine therapy. The prevailing dogma is that elevated circulating estrogen, produced by aromatase in abundant adipose tissue, drives breast cancer formation and progression. While estrogen is clearly an important factor in the obesity-breast cancer relationship, increasing the dose of aromatase inhibitors provides no added benefit to obese women with breast cancer. A switch from hormone- to growth factor-dependence has been implicated in the development of endocrine therapy resistance. Hypothesis and Study Objective: We hypothesized that obese, postmenopausal women have elevated growth factor production in adipose tissue, which supports the progression of breast tumors that were previously dependent on estrogen. The purpose of our study was to develop a transplant-competent mouse model of obesity and postmenopausal breast cancer using estrogen receptor (ER) positive breast cancer patient-derived xenografts (PDX) from peri- and postmenopausal women (ages 45-71). Methods: Rag1-null mice on the C57Bl/6 background were fed either a low fat (LFD) or high fat (HFD) diet for 10 weeks. Thermoneutral housing was used to facilitate development of the obese phenotype. Animal weights and percent body fat were monitored. After weight separation (approximately 10 weeks), mice were ovariectomized and implanted with an ER+ estrogen-dependent PDX derived from a 54 year old woman bilaterally into the orthotopic site (inguinal mammary fat pad). Mice were given slow release estrogen pellets and tumor growth was monitored. After 8 weeks of tumor growth, a subset of animals received estrogen withdrawal (EWD) to mimic the menopausal transition. Results: HFD-fed mice weighed more, and had significantly increased body fat percentages, as well as subcutaneous, and gonadal fat pad weights, compared to LFD-fed mice. After EWD, tumors regressed in LFD-fed mice; however, the HFD prevented tumor regression in the absence of estrogen. Circulating estrogen levels in both LFD and HFD EWD groups were not significantly different. Tumor cellularity and Ki67 were greater in HFD- compared to LFD-fed mice in the absence of estrogen. Phospho-protein analysis of tumors suggested increased human epidermal growth factor receptor 2 (HER2) and fibroblast growth factor receptor (FGFR) signaling in HFD-EWD versus LFD-EWD mice. Conclusions: We have developed a novel, transplant-competent mouse model of obesity and menopause using a specialized HFD and thermoneutral housing. Using this model, we determined that HFD supports the growth of a previously estrogen-dependent breast cancer PDX after EWD, potentially through increased signaling through HER2 and FGFR pathways. Our mouse model, in combination with PDX breast tumors, will offer novel insights into the relationships between obesity, menopause, and ER+ breast cancer progression. Citation Format: Elizabeth Wellberg, Britta Jacobsen, Peter Kabos, Paul MacLean, Carol Sartorius. A patient-derived xenograft model to study the effects of postmenopausal obesity on endocrine therapy resistance. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A08.

Abstract B133: In vivo imaging of innate immune cells to measure drug response

Abstract Background: Tumor-associated macrophages (TAMs) play key roles in the progression of cancers and their response to various treatments, for example through secretion of pro-survival growth factors; contribution to angiogenesis; and modulation of the tumor immune response via antigen presentation and expression of factors such as PD-L1. TAM effects can be highly localized within the tumor microenvironment, for example through cell-cell contact, yet it has been difficult to directly study interactions between TAMs and tumor cells at high resolution, in orthotopic sites of cancer development, and in real-time as cells respond to therapeutic treatment. To understand the biology underlying heterogeneous interactions between TAMs and tumor cells, we used in vivo fluorescence imaging at single-cell resolution to study pharmacodynamic (PD) response of tumor cells and TAMs to chemotherapeutic treatment. Methods: To directly image host leukocytes in a variety of tumor models, we used a combination of fluorescent genetic reporter host animals (CX3CR1GFP/+ reporter mice) and macrophage labeling via well-validated fluorescent and magnetic dextran-coated nanoparticles. For direct visualization of therapeutic response in cancer cells themselves, we used 53BP1-mApple as a transgenic fluorescent reporter to quantify DNA damage responses at the single-cell level. Automated computational segmentation of 3D images enabled precise quantification of statistical correlation between spatial TAM localization and cellular DNA damage responses. We performed studies in multiple human xenograft and syngeneic tumor models, including orthotopic ovarian cancer models and a syngeneic Kras-mutant p53-null lung adenocarcinoma model; histology validated in vivo imaging results. As a proof-of-principle, we tested cellular response to platinum (Pt)-based DNA-damaging agents. Results: Using a novel combination of orthotopic imaging setups, fluorescent reporters for TAMs and cancer cell pharmacodynamic response, along with automated 3D image segmentation and single-cell quantification, here we demonstrate that in vivo imaging can quantify correlations between local TAM concentrations and the simultaneously observed behavior of thousands of cancer cells. As a proof-of-principle, we measure relationships between single-cell DNA damage and local TAM levels in response to Pt-based chemotherapeutic treatment, and find that highly-localized concentrations of tumor-associated immune cells can influence therapeutic response at the single-cell level (and at cellular length-scale gradients). Importantly, we find these relationships to be highly probabilistic (rather than deterministic), and automated classification of thousands of cells across multiple tumors enables robust quantification of the inherent stochasticity involved in the pharmacodynamic responses. We find that single-cell heterogeneity and its explanation by microenvironmental factors such as local TAM concentration and vascular proximity can be heavily dependent on the therapeutic treatment and tumor model. Conclusions: This work presents in vivo imaging technology that should be useful for studying interactions between cancer cells and TAMs in orthotopic sites including intraperitoneal ovarian cancer and can be extended to other cell interactions (for instance by using fluorescent genetic reporters of various lymphocyte populations). Interaction and co-localization between tumor cells and associated immune cells can influence therapeutic response at the single-cell level, and such information will likely be useful for understanding heterogeneous drug responses to chemotherapeutics and immunotherapeutics alike. Citation Format: Miles A. Miller, Mikael Pittet, Ralph Weissleder. In vivo imaging of innate immune cells to measure drug response. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B133.

Demineralized dentin matrix combined with recombinant human bone morphogenetic protein-2 in rabbit calvarial defects.

ObjectivesThe aim of this study was to compare the osteogenic effects of demineralized dentin matrix (DDM) combined with recombinant human bone morphogenetic protein-2 (rhBMP-2) in rabbit calvarial defects with DDM and anorganic bovine bone (ABB) combined with rhBMP-2.Materials and MethodsFour round defects with 8-mm diameters were created in each rabbit calvaria. Each defect was treated with one of the following: 1) DDM, 2) ABB/rhBMP-2, or 3) DDM/rhBMP-2. The rhBMP-2 was combined with DDM and ABB according to a stepwise dry and dip lyophilizing protocol. Histological and microcomputed tomography (µCT) analyses were performed to measure the amount of bone formation and bone volume after 2- and 8-week healing intervals.ResultsUpon histological observation at two weeks, the DDM and ABB/rhBMP-2 groups showed osteoconductive bone formation, while the DDM/rhBMP-2 group showed osteoconductive and osteoinductive bone formation. New bone formation was higher in DDM/rhBMP-2, DDM and ABB decreasing order. The amounts of bone formation were very similar at two weeks; however, at eight weeks, the DDM/rhBMP-2 group showed a two-fold greater amount of bone formation compared to the DDM and ABB/rhBMP-2 groups. The µCT analysis showed markedly increased bone volume in the DDM/rhBMP-2 group at eight weeks compared with that of the DDM group. Notably, there was a slight decrease in bone volume in the ABB/rhBMP-2 group at eight weeks. There were no significant differences among the DDM, ABB/rhBMP-2, and DDM/rhBMP-2 groups at two or eight weeks.ConclusionWithin the limitations of this study, DDM appears to be a suitable carrier for rhBMP-2 in orthotopic sites.

Abstract A9: Bioluminescent orthotopic PDX models of primary pancreatic cancer and residual/metastatic breast cancer predict efficacy of standard of care and experimental treatments

Abstract Background: Only 5% of investigational anti-cancer agents are ultimately clinically successful. This may be attributed in part to the historical preclinical use of human cell lines that poorly recapitulate tumor heterogeneity in imperfect xenograft models. To model critical influences of tissue environment on tumor behavior and therapeutic response, heterogeneous patient-derived xenografts (PDX) can be assayed following implantation at orthotopic sites in rodents. Challenges to this approach include inaccessibility of certain organs or inability to track residual or metastatic disease. To address these problems, we utilized pancreatic (PANx-005) or metastatic breast (HBCx-14) PDXs transduced with stable bioluminescent reporters in efficacy studies of standard of care (SOC) and experimental treatments in clinically recapitulative models. Methods: Freshly excised PDXs were transduced with lentiviral vectors stably expressing luciferase, and implanted orthotopically into NOD scid gamma (NSG) mice. For our primary pancreatic cancer model, the pancreas was surgically exposed, and ∼2.5 × 106 PANx-005-Luc cells were inoculated directly. Post-op, tumor growth was monitored in-life (Xenogen IVIS® Lumina Series III instrument (IVIS)). Mice were randomized into treatment groups when mean tumor radiance (TR) reached 3.0 × 106 photons/sec. Control animals received no treatment; SOC groups received either focused fractionated radiation (12 Gy as 3×4 Gy fractions, Q5D (SRT)) or gemcitabine (75 mg/kg, 2QWx4, i.p.), and the experimental group received (+)-JQ-1 BET bromodomain inhibitor (50 mg/kg, QD, i.p.). For the residual/metastatic breast cancer model, ∼1.5 × 106 HBCx-14-Luc were injected directly into the 4th inguinal mammary fat pad. Tumor growth and response were monitored throughout the study by IVIS. When individual TR reached 7.5 × 109 photons/sec, the primary tumor was resected and the animal enrolled into a treatment group to be treated for residual/metastatic disease. Control animals received no treatment; SOC groups received either SRT, capacetabine (540 mg/kg QDx5/ 1week rest, p.o.) or docetaxel (20 mg/kg, Q3Wx2, i.p.); while experimental animals received (+)-JQ-1 (50 mg/kg, QD, i.p.). Results: Tumor seeding approached 100% in both models. Growth kinetics resembled clinical indications, including rapid growth and lung metastases for the HBCx-14 tumors, and slower growth of PANCx-005 tumors. Efficacies of individual treatments recapitulated clinical responses: HBCx-14 tumors were high responders to capecitabine and (+)-JQ-1, and low-responders to docetaxel, while PANx-005 tumors displayed high response to (+)-JQ-1 and lower response to gemcitabine. Conclusions: The translational predictivity of preclinical cancer models is enhanced by the use of human PDXs that preserve tumor heterogeneity, are assayed at orthotopic sites, and are utilized in models that recapitulate clinical situations (e.g. treatment of residual disease vs primary tumor). The use of stable bioluminescent reporters in such assays greatly enhances precision in monitoring tumor growth and treatment response. Currently available preclinical oncology models are more recapitulative, precise and predictive than ever before, and appear poised to engender improved translational success. Citation Format: Benjamin G. Cuiffo, Olivier Deas, Gregory D. Lyng, Stephano Cairo, Enora Le Ven, Jean-Gabriel Judde, Stephen T. Sonis. Bioluminescent orthotopic PDX models of primary pancreatic cancer and residual/metastatic breast cancer predict efficacy of standard of care and experimental treatments. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A9.

VSELs may obviate cryobanking of gonadal tissue in cancer patients for fertility preservation.

BackgroundInfertility is an undesirable side effect and gonadal tissue banking is advocated in young cancer patients who are unable to preserve embryos or gametes prior to oncotherapy to achieve biological parenthood later on. Banking gonadal tissue is challenging and protocols to mature gametes in vitro are not yet clinically established. Transplanting ovarian cortical tissue at hetero-or orthotopic sites in women and bone marrow transplantation (BMT) in both men and women has resulted in spontaneous recovery of fertility, pregnancy and live births. Various studies in humans and mice suggest that genetic origin of offspring after BMT is similar to transplanted patient and not the donor. Thus the source of oocytes/sperm which result in spontaneous pregnancies still remains contentious.FindingsVery small embryonic-like stem cells (VSELs) have been reported in adult human testis and ovary, in azoospermic testicular biopsies from survivors of childhood cancer and also in women with premature ovarian failure and menopause. VSELs survive chemotherapy because of their quiescent nature and can be detected in chemoablated mice gonads at protein and mRNA level and also by flow cytometry. Surviving VSELs spontaneously differentiate into oocyte-like structures and sperm when inhibitory factors are overcome in vitro. Transplantation of mesenchymal cells (isolated from different sources) has led to regeneration of chemoablated mouse gonads and also live births. Spermatogenesis is also restored from endogenous stem cells on inter-tubular transplantation of Sertoli cells in chemoablated mouse testis.ConclusionsEndogenous VSELs (which survive oncotherapy) can possibly regenerate non-functional gonads in cancer survivors when exposed to a healthy niche in vitro or in vivo (by way of transplanting mesenchymal cells which secrete trophic factors required for endogenous VSELs to differentiate into gametes). Presence of VSELs can also explain spontaneous pregnancies after BMT and cortical tissue transplantation (at heterotopic or orthotopic sites). This understanding once verified and accepted by the scientific community could obviate the need to remove whole ovary or testicular biopsy for cryopreservation prior to oncotherapy.

A Review of Current Animal Models for the Study of Cervical Dysplasia and Cervical Carcinoma.

Cancer research has long relied on animal models for the study of disease mechanisms and new therapeutics. Future cancer treatments are likely to rely heavily on patient-derived xenograft models to develop novel treatments and tailor regimens to individual patient needs. However, specific models for cervical cancer and cervical dysplasia are limited. Only 3 models have been described in the published literature. A transgenic model for cervical cancer has allowed for the study of the differential contributions of the human papillomavirus 16 proteins E6 and E7 during oncogenesis. This model has also shown dysplasia development, although this has received little attention. A patient-derived tumor xenograft model where cervical cancer tissue is transplanted to the subcutaneous and orthotopic sites has been described. Here we review the reported transgenic and xenograft models, their strengths and limitations, and highlight the potential for the development of improved models to study cervical neoplasia.

Longitudinal far red gene-reporter imaging of cancer metastasis in preclinical models: a tool for accelerating drug discovery.

In this short communication, we demonstrate for the first time, the use of far red fluorescent gene reporter, iRFP to longitudinally and non-invasively track the in vivo process of lymphatic metastases from an orthotopic site of mammary implantation through lymphatic vessels and to draining lymph nodes. Potentially useful to accelerate cancer drug discovery as an in vivo screening tool to monitor the pharmacological arrest of metastasis, we show that the custom as well as commercial small animal imaging devices have adequate performance to detect the gene reporter in stably expressing metastatic cancer cells.

Neoplastic transformation of porcine mammary epithelial cells in vitro and tumor formation in vivo.

BackgroundThe mammary glands of pigs share many functional and morphological similarities with the breasts of humans, raising the potential of their utility for research into the mechanisms underlying normal mammary function and breast carcinogenesis. Here we sought to establish a model for the efficient manipulation and transformation of porcine mammary epithelial cells (pMEC) in vitro and tumor growth in vivo.MethodsWe utilized a vector encoding the red florescent protein tdTomato to transduce populations of pMEC from Yorkshire –Hampshire crossbred female pigs in vitro and in vivo. Populations of primary pMEC were then separated by FACS using markers to distinguish epithelial cells (CD140a-) from stromal cells (CD140a+), with or without further enrichment for basal and luminal progenitor cells (CD49f+). These separated pMEC populations were transduced by lentivirus encoding murine polyomavirus T antigens (Tag) and tdTomato and engrafted to orthotopic or ectopic sites in immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice.ResultsWe demonstrated that lentivirus effectively transduces pMEC in vitro and in vivo. We further established that lentivirus can be used for oncogenic-transformation of pMEC ex vivo for generating mammary tumors in vivo. Oncogenic transformation was confirmed in vitro by anchorage-independent growth, increased cell proliferation, and expression of CDKN2A, cyclin A2 and p53 alongside decreased phosphorylation of Rb. Moreover, Tag-transformed CD140a- and CD140a-CD49f + pMECs developed site-specific tumors of differing histopathologies in vivo.ConclusionsHerein we establish a model for the transduction and oncogenic transformation of pMEC. This is the first report describing a porcine model of mammary epithelial cell tumorigenesis that can be applied to the study of human breast cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1572-7) contains supplementary material, which is available to authorized users.

Systemic delivery of micelles loading with paclitaxel using N-succinyl-palmitoyl-chitosan decorated with cRGDyK peptide to inhibit non-small-cell lung cancer

This study aimed to prepare efficient cRGDyK peptide-decorated micelles for the targeted therapy of non-small-cell lung cancer (NSCLC). An amphiphilic copolymer N-succinyl-palmitoyl-chitosan (SPCS) was synthesized and characterized. cRGDyK peptide is a ligand that can target tumors via specific binding integrin receptor overexpressed on tumor neovascularization and cells. cRGDyK-functionalized SPCS micelles loaded with paclitaxel (PTX/cRGDyK-SPCS) were prepared by film dispersion method and then characterized according to morphology, size, and zeta potential. PTX/cRGDyK-SPCS micelles presented pH-triggered drug release behavior under acidic conditions. The accumulation of micelles detected by laser confocal fluorescence microscopy and flow cytometry showed that cRGDyK-SPCS micelles were easily taken up by A549 cells marked with the luciferase gene (luc-A549). Meanwhile, co-localization of the micelles and lysosomes was recorded dynamically using a live cell station. MTT assays and cell apoptosis studies revealed that cell viability was significantly inhibited by PTX/cRGDyK-SPCS micelles. More importantly, in vivo animal studies showed that cRGDyK-SPCS micelles mainly accumulated in the orthotopic tumor site. PTX/cRGDyK-SPCS micelles exhibited better anti-tumor activity in subcutaneous and orthotopic lung tumors compared with PTX/SPCS micelles and Taxol®. These results suggested that PTX/cRGDyK-SPCS micelles had better cancer targeting capacity and superior anti-tumor efficacy. Thus, these micelles have great potential as novel carriers in delivering anti-tumor drugs.

Ovarian Cryopreservation and Transplantation: A Preserving Fertility Procedure

Cancer is a major health problem in both developed and developing countries. In women, cancer incidence rates increased every year. Developments in treatment modalities and the ability to detect tumours in the early stages increased their survival rate but also raise fertility problems. Those problems are the fertility preservation for patient who have to endure gonadotoxic chemotherapy and or radiation even though they still need their fertility functions. Ovarian cryopreservation and autotransplantation were initially designed to protect and restore reproductive function in patients receiving sterilizing chemotherapy and/or radiotherapy. Other indications including patients undergoing haematopietic stem cell transplantation, autoimmune diseases and those undergoing oophorectomy for non-cancer conditions. Options in cryopreserved ovarian tissues include autotransplantation and xenotransplantation. An orthotopic site or a heterotopic site can be considered for autotransplantation. Xenotransplantation of human ovarian tissue into immunodeficient animals can prevent immunological rejection. The theoretical advantage of orthotopic grafts is the restoration of normal reproductive function and natural conception after transplantation but application for cancer patients is problematic because of the potential risk of transmission of microscopic metastatic disease. With Xenotransplantantion, the possibility of cancer transmission and relapse can be eliminated because cancer cells cannot penetrate the zona pellucida, and some technical difficulties of in vitro growth and maturation of primordial follicles can be bypassed. But it is still unknown whether conditions for the growth and maturation of human oocytes in an animal host are comparable to those in situ and whether animal pathogens can be transmitted to human tissue with it. Ovarian tissue cryopreservation is the fertility preservation option for prepubertal girls and for women who face thehigh likelihood of diminished ovarian reserve requiring immediate treatment. Its procedure are still within improvement and also in the study of understanding its mechanism. In the future, studies and large clinical trials are still needed to develop better cryoprotectants and cryopreservation protocols and also standardization - optimization transplantation techniques

Therapeutic Effect on Bladder Cancer with aConditionally Replicating Oncolytic Virus Derived from Type II Herpes Simplex Virus.

Purpose:Despite recent improvements, resistance to traditional immunotherapy or chemotherapy is still common in patients with bladder cancer. We constructed an oncolytic virus from herpes simplex virus type II (HSV-2), which selectively targets tumor cells with an activated Ras signaling pathway. We evaluated the antitumor effect of this oncolytic HSV-2 (FusOn-H2) against bladder cancer, and compared with that of a first generation oncolytic virus derived from HSV-1 (Baco-1).Materials and methods: We established bladder tumor at the orthotopic site in C3H/He mice using the MBT-2 cells. Baco-1 or FusOn-H2 was instilled into the bladder through the urethra respectively. Tumor volume and weight were recorded by the end of the experiment. Animal spleens were also collected to determine if any anti-tumor immunity was elicited during virotherapy in this syngeneic bladder cancer model.Results: Two instillations of the oncolytic HSVs into bladder of tumor-bearing mice almost completely eradicated the tumor in majority of tumor bearing mice. The results of tumor-specific cytotoxic T lymphocyte activity assay showed that tumor destruction by oncolytic viruses in vivo, especially by the FusOn-H2, induced potent anti-tumor immune responses.Conclusion: Oncolytic virus derived from HSV-2 has potent anti-tumor activity against bladder cancer. Oncolytic effect of this virus in vivo induces tumor specific cellular immunity that further enhances the overall anti-tumor activity. Translating this novel virotherapy into the clinic could present an alternative intravesical therapy strategy for patients with bladder cancer.

Pre-clinical in vivo models for the screening of bone biomaterials for oral/craniofacial indications: focus on small-animal models.

Preclinical in vivo experimental studies are performed for evaluating proof-of-principle concepts, safety and possible unwanted reactions of candidate bone biomaterials before proceeding to clinical testing. Specifically, models involving small animals have been developed for screening bone biomaterials for their potential to enhance bone formation. No single model can completely recreate the anatomic, physiologic, biomechanic and functional environment of the human mouth and jaws. Relevant aspects regarding physiology, anatomy, dimensions and handling are discussed in this paper to elucidate the advantages and disadvantages of small-animal models. Model selection should be based not on the 'expertise' or capacities of the team, but rather on a scientifically solid rationale, and the animal model selected should reflect the question for which an answer is sought. The rationale for using heterotopic or orthotopic testing sites, and intraosseous, periosseous or extraskeletal defect models, is discussed. The paper also discusses the relevance of critical size defect modeling, with focus on calvarial defects in rodents. In addition, the rabbit sinus model and the capsule model in the rat mandible are presented and discussed in detail. All animal experiments should be designed with care and include sample-size and study-power calculations, thus allowing generation of meaningful data. Moreover, animal experiments are subject to ethical approval by the relevant authority. All procedures and the postoperative handling and care, including postoperative analgesics, should follow best practice.

MP45-01 HIGH ALDO-KETO REDUCTASE 1C1 EXPRESSION IN METASTATIC BLADDER CANCER CELLS ASSOCIATED WITH INVASIVE POTENTIAL AND DRUG RESISTANCE

You have accessJournal of UrologyBladder Cancer: Basic Research II1 Apr 2015MP45-01 HIGH ALDO-KETO REDUCTASE 1C1 EXPRESSION IN METASTATIC BLADDER CANCER CELLS ASSOCIATED WITH INVASIVE POTENTIAL AND DRUG RESISTANCE Ryuji Matsumoto, Masumi Tsuda, Takashige Abe, Satoru Maruyama, Kunihiko Tsuchiya, Naoto Miyajima, Nobuo Shinohara, and Shinya Tanaka Ryuji MatsumotoRyuji Matsumoto More articles by this author , Masumi TsudaMasumi Tsuda More articles by this author , Takashige AbeTakashige Abe More articles by this author , Satoru MaruyamaSatoru Maruyama More articles by this author , Kunihiko TsuchiyaKunihiko Tsuchiya More articles by this author , Naoto MiyajimaNaoto Miyajima More articles by this author , Nobuo ShinoharaNobuo Shinohara More articles by this author , and Shinya TanakaShinya Tanaka More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1499AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The objective of this study were to investigate the genetic changes responsible for bladder cancer metastasis, using orthotopic metastatic mouse model, and to find new therapeutic targets of invasive bladder cancer. METHODS Human UM-UC-3 bladder cancer cells were generated to express firefly luciferase 2 and red fluorescent protein tdTomato (UM-UC-3-tdTomato-luc2). Subsequently, we developed an orthotopic xenograft nude mouse model of bladder cancer by inoculating UM-UC-3-tdTomato-luc2 cells through a urethral catheter into the mouse bladder. We then performed bioluminescent imaging (BLI) to noninvasively monitor the growth of bladder tumors in their orthotopic sites. Two mice underwent autopsy after confirmation of lung or liver metastases using BLI. The primary and metastatic tumors were excised and plated on tissue culture dishes to generate bladder, lung, liver, and bone sublines. To examine the role of metastasis-related genes in bladder cancer, we then performed comparative microarray analyses of gene expression, and analyzed gene expression changes in bladder vs. lung, bladder vs. liver and bladder vs. bone. RESULTS Among the 8 genes commonly up-regulated in metastatic sublines compared to bladder cells by our microarray analysis, the expression of aldo-keto reductase 1C1 (AKR1C1) was especially up-regulated. Furthermore, AKR1C1 was significantly upregulated in the 3 metastatic sublines by qRT-PCR and immunoblot analysis. In clinical samples, we observed increased expression of AKR1C1 in human metastatic tissues compared with the corresponding primary bladder cancer tissues by qRT-PCR and immunohistochemistry analysis. Knockdown of AKR1C1 using small interfering RNA downregulated expression levels of Rac1, phosphorylated Src, and phosphorylated FAK, accompanied by reduced invasive ability. Overexpression of AKR1C isoforms, including AKR1C1, has been demonstrated to be associated with drug resistance in various cancers. UM-CU-3 metastatic cells were more resistant to cisplatin than were UM-UC-3 wild-type cells, and AKR1C1 inhibitor flufenamic acid reversed cisplatin resistance in these cells. CONCLUSIONS We found that AKR1C1 was up-regulated both in metastatic bladder cancer cells using an orthotopic mouse model and in metastatic sites of human surgical specimens. Our results demonstrate the role of AKR1C1 in regulating bladder cancer metastasis and drug resistance; therefore, AKR1C1 is a potential target for effective treatment of invasive bladder cancer. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e535 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ryuji Matsumoto More articles by this author Masumi Tsuda More articles by this author Takashige Abe More articles by this author Satoru Maruyama More articles by this author Kunihiko Tsuchiya More articles by this author Naoto Miyajima More articles by this author Nobuo Shinohara More articles by this author Shinya Tanaka More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

223 High aldo-keto reductase 1C1 expression in metastatic bladder cancer cells associated with invasive potential and drug resistance

INTRODUCTION AND OBJECTIVES: The objective of this study were to investigate the genetic changes responsible for bladder cancer metastasis, using orthotopic metastatic mouse model, and to find new therapeutic targets of invasive bladder cancer. METHODS: Human UM-UC-3 bladder cancer cells were generated to express firefly luciferase 2 and red fluorescent protein tdTomato (UM-UC-3-tdTomato-luc2). Subsequently, we developed an orthotopic xenograft nude mouse model of bladder cancer by inoculating UM-UC-3-tdTomato-luc2 cells through a urethral catheter into the mouse bladder. We then performed bioluminescent imaging (BLI) to noninvasively monitor the growth of bladder tumors in their orthotopic sites. Two mice underwent autopsy after confirmation of lung or liver metastases using BLI. The primary and metastatic tumors were excised and plated on tissue culture dishes to generate bladder, lung, liver, and bone sublines. To examine the role of metastasis-related genes in bladder cancer, we then performed comparative microarray analyses of gene expression, and analyzed gene expression changes in bladder vs. lung, bladder vs. liver and bladder vs. bone. RESULTS: Among the 8 genes commonly up-regulated in metastatic sublines compared to bladder cells by our microarray analysis, the expression of aldo-keto reductase 1C1 (AKR1C1) was especially up-regulated. Furthermore, AKR1C1 was significantly upregulated in the 3 metastatic sublines by qRT-PCR and immunoblot analysis. In clinical samples, we observed increased expression of AKR1C1 in human metastatic tissues compared with the corresponding primary bladder cancer tissues by qRT-PCR and immunohistochemistry analysis. Knockdown of AKR1C1 using small interfering RNA downregulated expression levels of Rac1, phosphorylated Src, and phosphorylated FAK, accompanied by reduced invasive ability. Overexpression of AKR1C isoforms, including AKR1C1, has been demonstrated to be associated with drug resistance in various cancers. UM-CU-3 metastatic cells were more resistant to cisplatin than were UM-UC-3 wild-type cells, and AKR1C1 inhibitor flufenamic acid reversed cisplatin resistance in these cells. CONCLUSIONS: We found that AKR1C1 was up-regulated both in metastatic bladder cancer cells using an orthotopic mouse model and in metastatic sites of human surgical specimens. Our results demonstrate the role of AKR1C1 in regulating bladder cancer metastasis and drug resistance; therefore, AKR1C1 is a potential target for effective treatment of invasive bladder cancer.