The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.
24 Related JoVE Articles!
Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation
Institutions: Virginia Commonwealth University- Massey Cancer Center, Virginia Commonwealth University- Massey Cancer Center, Virginia Commonwealth University- Massey Cancer Center.
It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2
. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo
for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3
. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4
. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo
expanded T cells is determined.
Immunology, Issue 47, Adoptive T cell therapy, Breast Cancer, HER-2/neu, common gamma chain cytokines, Bryostatin 1, Ionomycin
Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy
Institutions: Roswell Park Cancer Institute, Sunnybrook Research Institute.
One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.
Medicine, Issue 86, Spontaneous metastasis, orthotopic, nephrectomy, renal cell carcinoma, RCC, necropsy, kidney, bioluminescence, sub-capsular
Anterior Cervical Discectomy and Fusion in the Ovine Model
Institutions: Monash University, Monash University.
Anterior cervical discectomy and fusion (ACDF) is the most common surgical operation for cervical radiculopathy and/or myelopathy in patients who have failed conservative treatment1,5
. Since the operation was first described by Cloward2
and Smith and Robinson6
in 1958, a variety refinements in technique, graft material and implants have been made3
. In particular, there is a need for safe osteoinductive agents that could benefit selected patients. The ovine model has been shown to have anatomical, biomechanical, bone density and radiological properties that are similar to the human counterpart, the most similar level being C3/44
. It is therefore an ideal model in which preclinical studies can be performed. In particular this methodology may be useful to researchers interested in evaluating different devices and biologics, including stem cells, for potential application in human spinal surgery.
Medicine, Issue 32, Anterior cervical discectomy, interbody fusion, spine fusion, stem cells, biologics, spine instrumentation, interbody cage
Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples
Institutions: University of Manitoba, University of Manitoba.
Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo
flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.
Medicine, Issue 89, mucosal, immunology, FGT, lavage, cervical, CMC
Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia
Institutions: University of Wisconsin-Madison, University of Rochester School of Medicine & Dentistry, University of Wisconsin-Madison.
New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.
Medicine, Issue 78, Cancer Biology, Prostatic Hyperplasia, Prostatic Neoplasms, Neoplastic Processes, Estradiol, Testosterone, Transplantation, Heterologous, Growth, Xenotransplantation, Heterologous Transplantation, Hormones, Prostate, Testosterone, 17beta-Estradiol, Benign prostatic hyperplasia, Prostate Cancer, animal model
Models of Bone Metastasis
Institutions: Vanderbilt University, Vanderbilt University, Tennessee Valley Healthcare System (VISN 9), Vanderbilt University, Vanderbilt University.
Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1
. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo
models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2
, that has since been transfected for GFP expression and re-cloned by our group3
. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2
, in certain instances intratibial4
or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.
Medicine, Issue 67, Mouse models of bone metastasis, breast cancer, cancer biology, intracardiac injections, intratibial injections, tumor cells
A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor
Institutions: University of British Columbia , University of British Columbia .
While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1
Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5° (animals rotated at 22.5°), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.
Medicine, Issue 65, Neuroscience, Physiology, Infinite Horizon Spinal Cord Injury Device, SCI, cervical, unilateral, contusion, forelimb function
Assessing Forelimb Function after Unilateral Cervical SCI using Novel Tasks: Limb Step-alternation, Postural Instability and Pasta Handling
Institutions: The University of Texas at Austin, The University of Texas at Austin, University of Florida.
Cervical spinal cord injury (cSCI) can cause devastating neurological deficits, including impairment or loss of upper limb and hand function. A majority of the spinal cord injuries in humans occur at the cervical levels. Therefore, developing cervical injury models and developing relevant and sensitive behavioral tests is of great importance. Here we describe the use of a newly developed forelimb step-alternation test after cervical spinal cord injury in rats. In addition, we describe two behavioral tests that have not been used after spinal cord injury: a postural instability test (PIT), and a pasta-handling test. All three behavioral tests are highly sensitive to injury and are easy to use. Therefore, we feel that these behavioral tests can be instrumental in investigating therapeutic strategies after cSCI.
Behavior, Issue 79, Behavior, Animal, Motor Activity, Nervous System Diseases, Wounds and Injuries, cervical spinal cord injury, lateral hemisection model, limb alternation, pasta handling, postural instability
A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity
Institutions: Université de Versailles Saint-Quentin-en-Yvelines, Hôpital Ambroise Paré, Université de Versailles Saint-Quentin-en-Yvelines.
A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.
Physiology, Issue 87, rat, cervical spinal cord injury, respiratory deficit, crossed phrenic phenomenon, respiratory neuroplasticity
Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury
Institutions: Thomas Jefferson University Medical College.
Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1
and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3
. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4
. Transgenic mice 4,5
and rats 6
carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R)
have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7
. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1
. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11
. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A
mice and rats, as well as spinal cord injured rats and mice 11
Medicine, Issue 55, cell transplantation, engraftment, graft, spinal cord, stem cells, precursors, ALS, amyotrophic lateral sclerosis, motor neuron, SCI, spinal cord injury
Murine Bioluminescent Hepatic Tumour Model
Institutions: University College Cork, University College Cork, South Infirmary Victoria University Hospital.
This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis.
JoVE Medicine, Issue 41, Cancer, Therapy, Liver, Orthotopic, Metastasis
Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology
Institutions: University Medical Center Groningen, Technical University Munich, University Medical Center Groningen.
The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3
Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5
As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6
In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7
The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8
It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.
Medicine, Issue 44, Image-guided surgery, multispectral fluorescence, sentinel lymph node, gynecologic oncology
Induction and Analysis of Epithelial to Mesenchymal Transition
Institutions: R&D Systems, Inc., R&D Systems, Inc..
Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro
is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.
Molecular Biology, Issue 78, Cellular Biology, Biochemistry, Biomedical Engineering, Stem Cell Biology, Cancer Biology, Medicine, Bioengineering, Anatomy, Physiology, biology (general), Pathological Conditions, Signs and Symptoms, Wounds and Injuries, Neoplasms, Diagnosis, Therapeutics, Epithelial to mesenchymal transition, EMT, cancer, metastasis, cancer stem cell, cell, assay, immunohistochemistry
Substernal Thyroid Biopsy Using Endobronchial Ultrasound-guided Transbronchial Needle Aspiration
Institutions: State University of New York, Buffalo, Roswell Park Cancer Institute, State University of New York, Buffalo.
Substernal thyroid goiter (STG) represents about 5.8% of all mediastinal lesions1
. There is a wide variation in the published incidence rates due to the lack of a standardized definition for STG. Biopsy is often required to differentiate benign from malignant lesions. Unlike cervical thyroid, the overlying sternum precludes ultrasound-guided percutaneous fine needle aspiration of STG. Consequently, surgical mediastinoscopy is performed in the majority of cases, causing significant procedure related morbidity and cost to healthcare. Endobronchial Ultrasound-guided Transbronchial Needle Aspiration (EBUS-TBNA) is a frequently used procedure for diagnosis and staging of non-small cell lung cancer (NSCLC). Minimally invasive needle biopsy for lesions adjacent to the airways can be performed under real-time ultrasound guidance using EBUS. Its safety and efficacy is well established with over 90% sensitivity and specificity. The ability to perform EBUS as an outpatient procedure with same-day discharges offers distinct morbidity and financial advantages over surgery. As physicians performing EBUS gained procedural expertise, they have attempted to diversify its role in the diagnosis of non-lymph node thoracic pathologies. We propose here a role for EBUS-TBNA in the diagnosis of substernal thyroid lesions, along with a step-by-step protocol for the procedure.
Medicine, Issue 93, substernal thyroid, retrosternal thyroid, intra-thoracic thyroid, goiter, endobronchial ultrasound, EBUS, transbronchial needle aspiration, TBNA, biopsy, needle biopsy
Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer
Institutions: Wistar Institute, University of Pennsylvania, Geisel School of Medicine at Dartmouth, University of Pennsylvania, University of Pennsylvania, University of Pennsylvania.
Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of αβ and γδ T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.
Medicine, Issue 85, Transgenic mice, breast cancer, metastasis, intraductal injection, latent mutations, adenovirus-Cre
Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice: A Model for Immunotherapy Development
Institutions: University of California, Davis, University of California, Davis, Merck KGaA, Darmstadt, Germany.
A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-γ)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.
Medicine, Issue 80, Urinary Bladder, Animals, Genetically Modified, Cancer Vaccines, Immunotherapy, Animal Experimentation, Models, Neoplasms Bladder Cancer, C57BL/6 Mouse, MUC1, Immunotherapy, Preclinical Model
Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres
Institutions: George Mason University, Virginia Surgery Associates.
Breast ductal carcinoma in situ
(DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2
incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.
Cancer Biology, Issue 93, Breast, ductal carcinoma in situ, epidermal growth factor, mammosphere, organoid, pre-invasive, primary cell culture, serum-free, spheroid
An Orthotopic Murine Model of Human Prostate Cancer Metastasis
Institutions: Northwestern University, Northwestern University, Northwestern University.
Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.
Medicine, Issue 79, Urogenital System, Male Urogenital Diseases, Surgical Procedures, Operative, Life Sciences (General), Prostate Cancer, Metastasis, Mouse Model, Drug Discovery, Molecular Biology
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells
Institutions: University of Michigan Comprehensive Cancer Center, University of Michigan Comprehensive Cancer Center.
Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1
. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex vivo
. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4
. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3
. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7
. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.
Medicine, Issue 86, pathological conditions, signs and symptoms, neoplasms, three dimensional cultures, Matrigel, breast cells, malignant phenotype, signaling
Analysis of Cell Migration within a Three-dimensional Collagen Matrix
Institutions: Witten/Herdecke University.
The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.
Bioengineering, Issue 92, cell migration, 3D collagen matrix, cell tracking
A Novel Method for Assessing Proximal and Distal Forelimb Function in the Rat: the Irvine, Beatties and Bresnahan (IBB) Forelimb Scale
Institutions: University of California, San Francisco.
Several experimental models of cervical spinal cord injury (SCI) have been developed recently to assess the consequences of damage to this level of the spinal cord (Pearse et al.
, 2005, Gensel et al.
, 2006, Anderson et al.
, 2009), as the majority of human SCI occur here (Young, 2010; www.sci-info-pages.com). Behavioral deficits include loss of forelimb function due to damage to the white matter affecting both descending motor and ascending sensory systems, and to the gray matter containing the segmental circuitry for processing sensory input and motor output for the forelimb. Additionally, a key priority for human patients with cervical SCI is restoration of hand/arm function (Anderson, 2004). Thus, outcome measures that assess both proximal and distal forelimb function are needed. Although there are several behavioral assays that are sensitive to different aspects of forelimb recovery in experimental models of cervical SCI (Girgis et al.
, 2007, Gensel et al.
, 2006, Ballerman et al.
, 2001, Metz and Whishaw, 2000, Bertelli and Mira, 1993, Montoya et al.
, 1991, Whishaw and Pellis, 1990), few techniques provide detailed information on the recovery of fine motor control and digit movement.
The current measurement technique, the Irvine, Beatties and Bresnahan forelimb scale (IBB), can detect recovery of both proximal and distal forelimb function including digit movements during a naturally occurring behavior that does not require extensive training or deprivation to enhance motivation. The IBB was generated by observing recovery after a unilateral C6 SCI, and involves video recording of animals eating two differently shaped cereals (spherical and doughnut) of a consistent size. These videos were then used to assess features of forelimb use, such as joint position, object support, digit movement and grasping technique.
The IBB, like other forelimb behavioral tasks, shows a consistent pattern of recovery that is sensitive to injury severity. Furthermore, the IBB scale could be used to assess recovery following other types of injury that impact normal forelimb function.
Neuroscience, Issue 46, spinal cord injury, recovery of function, forelimb function, neurological test, cervical injuries
Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice
Institutions: University of California, Irvine (UCI), University of California, Irvine (UCI).
Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response 1
. Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 2
. Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37°C.
Issue 7, Immunology, T Lymphocytes, Lymph Node, 2-photon Imaging, Tail Vein Injections
Right Hemihepatectomy by Suprahilar Intrahepatic Transection of the Right Hemipedicle using a Vascular Stapler
Institutions: Tübingen University Hospital.
Successful hepatic resection requires profound anatomical knowledge and delicate surgical technique. Hemihepatectomies are mostly performed after preparing the extrahepatic hilar structures within the hepatoduodenal ligament, even in benign tumours or liver metastasis.1-5
. Regional extrahepatic lymphadenectomy is an oncological standard in hilar cholangiocarcinoma, intrahepatic cholangio-cellular carcinoma and hepatocellular carcinoma, whereas lymph node metastases in the hepatic hilus in patients with liver metastasis are rarely occult. Major disadvantages of these procedures are the complex preparation of the hilus with the risk of injuring contralateral structures and the possibility of bleeding from portal vein side-branches or impaired perfusion of bile ducts. We developed a technique of right hemihepatectomy or resection of the left lateral segments with intrahepatic transection of the pedicle that leaves the hepatoduodenal ligament completely untouched. 6
However, if intraoperative visualization or palpation of the ligament is suspicious for tumor infiltration or lymph node metastasis, the hilus should be explored and a lymphadenectomy performed.
Medicine, Issue 35, Liver resection, liver tumour, intrahepatic hilus stapling, right hemipedicle