In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.Open in a separate windowClick here to view.^{(48M, flv)}     

Nude mice--a model system for studying the cellular basis of the humoral immune response

Mice homozygous for the nu gene fail to develop a thymus. In comparison to spleen cells from +/nu mice spleen cells from nu/nu mice have a deficient 19S PFC response to SRBC when tested in culture or in vivo. This deficiency is due to a lack of “helper” T cells in nu/nu spleen; A cells and B cells appear to be normal. The capacity of nu/nu spleen cells to produce a PFC response in culture can be corrected by the addition of T cells obtained from either the thymuses or the spleens of +/nu mice. In contrast to “helper” T cells obtained from the spleen, “helper” T cells obtained from the thymus appear to require the capacity for proliferation during the response to SRBC.     

Establishment of a Non-Invasive Semi-Quantitative Bioluminescent Imaging Method for Monitoring of an Orthotopic Esophageal Cancer Mouse Model

Orthotopic models of various types of tumors are widely used in anti-tumor therapeutic experiments in preclinical studies. However, there are few ways to appropriately monitor therapeutic effect in orthotopic tumor models, especially for tumors invisible from the outside. In this study we aimed to establish a non-invasive semi-quantitative bioluminescent imaging method of monitoring an orthotopic esophageal cancer mouse model. We confirmed that the TE8 esophageal cancer cell line implanted orthotopically into the abdominal esophagus of nu/nu mice (n = 5) developed not only a main tumor at the implanted site, but also local lymph node metastases and peritoneal disseminations within 6 weeks after inoculation. We established a TE8 cell line that stably expressed the firefly luciferase gene (TE8-Luc). We showed that TE8-Luc cells implanted subcutaneously into nu/nu mice (n = 5) grew over time until 5 weeks after inoculation. Tumor volume was strongly correlated with luminescent intensity emitted from the tumor, which was quantified using the IVIS imaging system. We then showed that TE8-Luc cells implanted orthotopically into the mouse abdominal esophagus (n = 8) also formed a tumor and that the luminescent intensity of such a tumor, as detected by IVIS, increased over time until 7 weeks after inoculation and was therefore likely to reflect tumor progression. We therefore propose that this orthotopic esophageal cancer model, monitored using the non-invasive semi-quantitative IVIS imaging system, will be useful for in vivo therapeutic experiments against esophageal cancer. This experimental setting is expected to contribute to the development of novel therapeutic technologies for esophageal cancer in preclinical studies.     

Thymus-independent induction and antigen-dependent recovery of idiotype-specific suppression

BALB/c nude (nu/nu) mice and euthymic (nu/+) littermates were treated as neonates with anti-T15 antibody and challenged at various ages with either a thymus-independent, PC-Brucella abortus (PC-BA), or thymus-dependent, PC-keyhole limpet hemocyanin (PC-KLH), form of phosphorylcholine (PC). Nu/nu mice challenged with PC-KLH received KLH-primed splenic T cells prior to immunization. Neither neonatally anti-idiotype-treated nu/+ nor nu/nu mice responded with the production of T15-positive anti-PC antibodies after challenge with either form of PC antigen. It is concluded that neither induction nor maintenance of a state of T15-specific suppression requires thymus-matured T cells. Recovery of anti-PC responsiveness in suppressed nu/+ or nu/nu mice was similar and was found to be related to the form of antigen used to elicit the response. Immunization with PC-KLH revealed a long-lasting unresponsiveness (up to 16 weeks). In contrast, immunization with PC-BA elicited a full anti-PC response as early as at 6.5 weeks of age.     

Lymphocyte function in experimental African trypanosomiasis. IV. Immunosuppression and suppressor cells in the athymic nu/nu mouse

The role of T cells in the development and expression of antigen-nonspecific immunosuppression in experimental African trypanosomiasis was addressed. Nude ($\text{nu}\text{nu}$) C57BL/ 6 NIH mice and their thymus-bearing ($\text{nu}\text{+}$) littermates were infected with Trypanosoma rhodesiense and examined for suppression of splenic B-cell responses in vitro to the mitogen LPS. All animals developed splenic unresponsiveness to LPS. Further, both nu/nu and nu/ + infected mice displayed suppressor cell activity in their spleen cell populations upon transfer to normal uninfected mouse spleen cell cultures. On the basis of these findings we suggest that both the generalized immunosuppression and the development of suppressor cell activity in the spleens of mice infected with T. rhodesiense are T-independent processes.     

Non-Invasive Bioluminescence Imaging to Monitor the Immunological Control of a Plasmablastic Lymphoma-Like B Cell Neoplasia after Hematopoietic Cell Transplantation

To promote cancer research and to develop innovative therapies, refined pre-clinical mouse tumor models that mimic the actual disease in humans are of dire need. A number of neoplasms along the B cell lineage are commonly initiated by a translocation recombining c-myc with the immunoglobulin heavy-chain gene locus. The translocation is modeled in the C.129S1-Igha^tm1(Myc)Janz/J mouse which has been previously engineered to express c-myc under the control of the endogenous IgH promoter. This transgenic mouse exhibits B cell hyperplasia and develops diverse B cell tumors. We have isolated tumor cells from the spleen of a C.129S1-Igha^tm1(Myc)Janz/J mouse that spontaneously developed a plasmablastic lymphoma-like disease. These cells were cultured, transduced to express eGFP and firefly luciferase, and gave rise to a highly aggressive, transplantable B cell lymphoma cell line, termed IM380. This model bears several advantages over other models as it is genetically induced and mimics the translocation that is detectable in a number of human B cell lymphomas. The growth of the tumor cells, their dissemination, and response to treatment within immunocompetent hosts can be imaged non-invasively in vivo due to their expression of firefly luciferase. IM380 cells are radioresistant in vivo and mice with established tumors can be allogeneically transplanted to analyze graft-versus-tumor effects of transplanted T cells. Allogeneic hematopoietic stem cell transplantation of tumor-bearing mice results in prolonged survival. These traits make the IM380 model very valuable for the study of B cell lymphoma pathophysiology and for the development of innovative cancer therapies.     

Application of 3.0 Tesla Magnetic Resonance Imaging for Diagnosis in the Orthotopic Nude Mouse Model of Pancreatic Cancer

The aim of this study was to successfully establish an orthotopic murine model using two different human pancreatic adenocarcinoma cell lines and to propose a 3.0 tesla MRI protocol for noninvasive characterization of this model. SW1990 and MIAPaca-2 tumor cells were injected into the pancreas of BALB/C nu/nu mice. Tumor growth rate and morphological information were assessed by 3.0 tesla MRI (T1WI, T2WI and DCE-MRI) and immunohistology. Proliferation of SW1990 was significantly faster than that of MIAPaca-2 (P=0.000), but MIAPaca-2 mice had a significantly shorter survival than SW1990 mice (41 days and 44 days respectively, P=0.027). MRI could reliably monitor tumor growth in both cell lines: the tumors exhibiting a spherical growth pattern showed a high-intensity signal, and the SW1990 group developed significantly larger tumors compared with the MIAPaCa-2 group. There were no statistical differences between the two groups in which tumor size was assessed using electronic calipers and an MRI scan (P=0.680). Both tumors showed a slow gradual enhancement pattern. Immunohistochemistry demonstrated tumor tissues showing high expression of Ki-67. This model closely mimics human pancreatic cancer and permits monitoring of tumor growth and morphological information by noninvasive 3.0 tesla MRI studies reducing the number of mice required.     

The Influence of the Athymic Mutation Nude on the Components of the Orcadian Rhythm of Activity in Mice

The mutation known as nude brings about the lack of a thymus gland in mice. This immuno-deficiency makes it possible to graft normally unaccepted, human cancerous tumors onto the mouse. Consequently, this animal is frequently used as a model for evaluating anti-cancer therapies. The effect of this mutation on biological rhythms constitutes a necessary step before using this model for cancer chronotherapy research. We evaluated the circadian and ultradian components of the restactivity cycle in the following strains of mice: CS7BL/6 with homozygous nu/nu, heterozygous nu/+, thymectomised +/+, and sham-operated +/+. The amount of activity was reduced in nu/nu as compared to the other groups. Nonetheless, neither the nude mutation nor thymectomy yielded any notable change in the circadian rhythm of activity.     

Anti-Tumor Effects after Adoptive Transfer of IL-12 Transposon-Modified Murine Splenocytes in the OT-I-Melanoma Mouse Model

Adoptive transfer of gene modified T cells provides possible immunotherapy for patients with cancers refractory to other treatments. We have previously used the non-viral piggyBac transposon system to gene modify human T cells for potential immunotherapy. However, these previous studies utilized adoptive transfer of modified human T cells to target cancer xenografts in highly immunodeficient (NOD-SCID) mice that do not recapitulate an intact immune system. Currently, only viral vectors have shown efficacy in permanently gene-modifying mouse T cells for immunotherapy applications. Therefore, we sought to determine if piggyBac could effectively gene modify mouse T cells to target cancer cells in a mouse cancer model. We first demonstrated that we could gene modify cells to express murine interleukin-12 (p35/p40 mIL-12), a transgene with proven efficacy in melanoma immunotherapy. The OT-I melanoma mouse model provides a well-established T cell mediated immune response to ovalbumin (OVA) positive B16 melanoma cells. B16/OVA melanoma cells were implanted in wild type C57Bl6 mice. Mouse splenocytes were isolated from C57Bl6 OT-I mice and were gene modified using piggyBac to express luciferase. Adoptive transfer of luciferase-modified OT-I splenocytes demonstrated homing to B16/OVA melanoma tumors in vivo. We next gene-modified OT-I cells to express mIL-12. Adoptive transfer of mIL-12-modified mouse OT-I splenocytes delayed B16/OVA melanoma tumor growth in vivo compared to control OT-I splenocytes and improved mouse survival. Our results demonstrate that the piggyBac transposon system can be used to gene modify splenocytes and mouse T cells for evaluating adoptive immunotherapy strategies in immunocompetent mouse tumor models that may more directly mimic immunotherapy applications in humans.     

The pathogenesis of trehalose dimycolate-induced interstitial pneumonitis: III. Evidence for a role for T lymphocytes

Trehalose dimycolate, a glycolipid component of the cell walls of mycobacteria, induces interstitial pneumonitis and alveolar hemorrhages in C57BL/6 and C57BL/10 mice. Homozygous nude (nu/nu) mice of these backgrounds are not susceptible to this form of pulmonary injury. However, after administration of T-lymphocyte-enriched spleen cell preparations from syngeneic donors, homozygous nude mice become susceptible to trehalose dimycolate. The observations suggest that production of pulmonary lesions by this mycobacterial component is dependent on T lymphocytes. While the mechanisms are still under study, we propose that trehalose dimycolate can function as an activator of T lymphocytes and that products of activated T cells are responsible for production of the pulmonary lesions.     

Mice Lacking NCF1 Exhibit Reduced Growth of Implanted Melanoma and Carcinoma Tumors

The NADPH oxidase 2 (NOX2) complex is a professional producer of reactive oxygen species (ROS) and is mainly expressed in phagocytes. While the activity of the NOX2 complex is essential for immunity against pathogens and protection against autoimmunity, its role in the development of malignant tumors remains unclear. We compared wild type and Ncf1 ^m1J mutated mice, which lack functional NOX2 complex, in four different tumor models. Ncf1 ^m1J mutated mice developed significantly smaller tumors in two melanoma models in which B16 melanoma cells expressing a hematopoietic growth factor FLT3L or luciferase reporter were used. Ncf1 ^m1J mutated mice developed significantly fewer Lewis Lung Carcinoma (LLC) tumors, but the tumors that did develop, grew at a pace that was similar to the wild type mice. In the spontaneously arising prostate carcinoma model (TRAMP), tumor growth was not affected. The lack of ROS-mediated protection against tumor growth was associated with increased production of immunity-associated cytokines. A significant increase in Th2 associated cytokines was observed in the LLC model. Our present data show that ROS regulate rejection of the antigenic B16-luc and LLC tumors, whereas the data do not support a role for ROS in growth of intrinsically generated tumors.     

Enhancement by interferon of natural killer cell activity in mice.

Injection of mice with several interferon inducers, Newcastle Disease virus, polyinosinic-polycytidylic acid and tilorone resulted in an increase in spleen cell cytotoxicity for ⁵¹chromium-labeled mouse YAC tumor target cells in 4-hr in vitro assays. This increase in spleen cell cytotoxicity was abrogated by injection of mice with potent anti-mouse interferon globulin. Inoculation of mice with mouse interferon (but not human leucocyte or mock interferon preparations) also resulted in a marked enhancement of spleen cell cytotoxicity. The extent of enhancement of spleen cell cytotoxicity was directly proportional to the amount of interferon injected and a significant increase was observed after inoculation of as little as 10³ to 10⁴ units of interferon. An effect could be detected as soon as 1 hr after injection of interferon. The increase of spleen cell cytotoxicity after inoculation of an interferon inducer was not due to a localization and accumulation of cytotoxic cells in the spleen but reflected a general increase in cytotoxic cell activity in various lymphoid tissues (except the thymus). The splenic cytotoxic cells from interferon or interferon-inducer-injected mice had the characteristics of natural killer (NK) cells since (i) interferon enhanced spleen cell cytotoxicity in athymic (nu/nu) nude mice, (ii) classical spleen cell fractionation procedures by nylon wool columns, anti-Thy 1.2 serum plus complement, anti-Ig columns, and depletion of FcR+ rosette-forming cells, failed to remove the effector cells generated in vivo or in vitro. Therefore like NK cells, interferon-induced cytotoxic cells lack the surface markers of mature T and B lymphocytes, are not adherent, and are devoid of avid Fc receptors. Furthermore like NK cells, the spleen cells from interferon-treated mice lysed various target cells (known for their sensitivity to NK cells) without H-2 or species restriction. Incubation in vitro of normal spleen cells with interferon also resulted in an increase in cytotoxicity for YAC tumor cells. We conclude that interferon acts directly on NK cells and enhances the inherent cytotoxic activity of these cells.     

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1^nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.     

Subclass restriction of murine antibodies: V. The IgG plaque-forming cell response to thymus-independent and thymus-dependent antigens in athymic and euthymic mice

Antigens differ in their abilities to stimulate antibodies of various isotypes. Many thymus-independent (TI) polysaccharide antigens stimulate largely IgG3 and IgM antibodies while thymus-dependent (TD) protein antigens stimulate predominantly IgG1 and smaller amounts of other isotypes. Here we determine whether thymus dependence or independence is a property of antigens which is expressed equally by all isotypes. To do this nu/+ and nu/nu mice were immunized with several TI and TD antigens and antibody responses of IgM and the four IgG subclasses measured. We found that, within the conditions of these experiments, all IgG isotypes were influenced equally by the presence or absence of T lymphocytes. Second, in agreement with J. L. Press (J. Immunol.126, 1234, 1981), we propose a division of TD antigens into two types based upon the ability to stimulate responses in the CBA/N mouse.     

Studies on immune responses to parasite antigens in mice: VII. Cells secreting antibodies to modified mouse erythrocytes in Babesia rodhaini-infected mice

Increased numbers of plaque-forming cells (PFC) secreting antibodies to bromelain-treated mouse erythrocytes were detected in the spleens of BALB/c·nu/+ mice heavily infected with Babesia rodhaini but not in the spleens of infected hypothymic BALB/ c·nu/nu mice. Whether the products of these cells, antierythrocyte autoantibodies, play any role in the destruction of intact erythrocytes in parasitized mice is unknown, but it is noteworthy that only low numbers of PFC were detected, and the modification of mouse erythrocytes was obligatory for detection of PFC.     

Low Dose Focused Ultrasound Induces Enhanced Tumor Accumulation of Natural Killer Cells

Natural killer (NK) cells play a vital antitumor role as part of the innate immune system. Efficacy of adoptive transfer of NK cells depends on their ability to recognize and target tumors. We investigated whether low dose focused ultrasound with microbubbles (ldbFUS) could facilitate the targeting and accumulation of NK cells in a mouse xenograft of human colorectal adenocarcinoma (carcinoembryonic antigen (CEA)-expressing LS-174T implanted in NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice) in the presence of an anti-CEA immunocytokine (ICK), hT84.66/M5A-IL-2 (M5A-IL-2). Human NK cells were labeled with an FDA-approved ultra-small superparamagnetic iron oxide particle, ferumoxytol. Simultaneous with the intravenous injection of microbubbles, focused ultrasound was applied to the tumor. In vivo longitudinal magnetic resonance imaging (MRI) identified enhanced accumulation of NK cells in the ensonified tumor, which was validated by endpoint histology. Significant accumulation of NK cells was observed up to 24 hrs at the tumor site when ensonified with 0.50 MPa peak acoustic pressure ldbFUS, whereas tumors treated with at 0.25 MPa showed no detectable NK cell accumulation. These clinically translatable results show that ldbFUS of the tumor mass can potentiate tumor homing of NK cells that can be evaluated non-invasively using MRI.     

Characterization of a Novel Anti-Cancer Compound for Astrocytomas

The standard chemotherapy for brain tumors is temozolomide (TMZ), however, as many as 50% of brain tumors are reportedly TMZ resistant leaving patients without a chemotherapeutic option. We performed serial screening of TMZ resistant astrocytoma cell lines, and identified compounds that are cytotoxic to these cells. The most cytotoxic compound was an analog of thiobarbituric acid that we refer to as CC-I. There is a dose-dependent cytotoxic effect of CC-I in TMZ resistant astrocytoma cells. Cell death appears to occur via apoptosis. Following CC-I exposure, there was an increase in astrocytoma cells in the S and G2/M phases. In in vivo athymic (nu/nu) nude mice subcutaneous and intracranial tumor models, CC-I completely inhibited tumor growth without liver or kidney toxicity. Molecular modeling and enzyme activity assays indicate that CC-I selectively inhibits topoisomerase IIα similar to other drugs in its class, but its cytotoxic effects on astrocytoma cells are stronger than these compounds. The cytotoxic effect of CC-I is stronger in cells expressing unmethylated O⁶-methylguanine methyltransferase (MGMT) but is still toxic to cells with methylated MGMT. CC-I can also enhance the toxic effect of TMZ on astrocytoma when the two compounds are combined. In conclusion, we have identified a compound that is effective against astrocytomas including TMZ resistant astrocytomas in both cell culture and in vivo brain tumor models. The enhanced cytotoxicity of CC-I and the safety profile of this family of drugs could provide an interesting tool for broader evaluation against brain tumors.     

Intracranial implantation with subsequent 3D in vivo bioluminescent imaging of murine gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain (1); therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation (1). Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1 mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4 mm to a depth of 2.6 mm. Two μl of GL261-luc or GL261-luc2 cells (10(7) cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured. Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150 μg luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in >10% loss of weight. Tumors were evident in all of the animals on necropsy.