Transgenic Nude Mice Research Articles

Imaging Transgenic Nude Mice Expressing Spectrally-distinct Fluorescent Reporters Emitting From Blue to Far Red Light With One Instrument With Single-nanometer-tuning of Laser-excitation Fluorescence.

Transgenic nude mice expressing green fluorescent protein (GFP), red fluorescent protein (RFP), or cyan fluorescent protein (CFP) were previously developed by our laboratory, AntiCancer Inc. In the present study, we demonstrate imaging of the GFP, RFP, or CFP nude mice with single-nanometer-tuning laser fluorescence excitation with a single instrument. Female transgenic C57/B6 nude GFP, RFP, and CFP mice aged six weeks were used. The images were obtained using the UVP Biospectrum Advanced system (Analytik Jena US LLC) with excitation at 480 nm and peak emission at 513 nm for GFP; 520 nm and 605 nm, respectively, for RFP; and 405 nm and 480 nm, respectively, for CFP. For each color transgenic fluorescent mouse, images without background could be obtained individually with the UVP Biospectrum Advanced system. Using a single instrument, brilliant and well-defined images of GFP, RFP, and CFP mice were obtained with single-nanometer-tuning laser fluorescence excitation. This imaging system will be used in future studies to analyze cancer cells in the colored mice that are spectrally distinct in order to determine how stromal cells and cancer interact in the tumor microenvironment.

Abstract P3-08-06: Monitoring Lymphovascular Invasion and Tumor Growth of Inflammatory Breast Cancer in a Murine Lymphatic Reporter Window Chamber Model

Abstract Background: Lymphovascular invasion (LVI) is a major route of metastatic dissemination and recent studies indicate its value as an independent prognostic indicator for advanced breast, colorectal, squamous cell, prostate, brain cancers. LVI is a clinicopathological hallmark of inflammatory breast cancer (IBC), an understudied and most lethal breast cancer. IBC is often misdiagnosed due to an absence of a solid mass and its unique presentation of diffuse tumor cell clusters/emboli in the dermal lymphatics. Widely used mammary tumor implantation models coupled with bioluminescence or fluorescence imaging to monitor tumor growth kinetics are ineffective for evaluating spatial and temporal changes in growth and migration patterns of individual tumor cells and clusters within their microenvironmental context. The goal of this study was to develop a murine model to simulate the unique clinicopathological features of IBC patients and to assess both qualitatively and quantitatively local tumor growth, motility, and LVI. Methods: To specifically facilitate visualization of lymphatic and endothelial vessels along with tumor-vessel interactions, we generated a transgenic nude mice model (ProxTom RFP Nu/Nu) wherein, the mice exhibit red, fluorescent lymphatics [tdTomato fluorophore under control of a Prox1 promoter, which encodes a transcription factor (prospero-related homeobox 1) necessary for the formation and maintenance of lymphatic vessels]. Next, we employed a surgical technique, wherein a window chamber is placed on the dorsal skinfold of mice, which allows for microscopic examination of implanted tumor cells and ability to track dynamic changes of the tumor in its local microenvironment from the time of implantation up to 10 days. Patient-derived IBC or PDX stably transfected to express green, red fluorescent and/or dual tagged with luciferase reporters were transplanted in mice bearing window chambers. Intravital fluorescence microscopy and IVIS imaging were used to serially quantify local tumor growth, motility, length density of lymph and blood vessels, and degree of tumor cell lymphatic invasion over 0-140h. Results: Multichannel optical imaging of the window chamber in the ProxTom RFP Nu/Nu mice demonstrated co-localization of IBC tumor cells and lymphatics. Diffuse tumor cells were observed along regions of lymphatic vessels both proximal and distal to the primary tumor site. However, measurement of blood and lymph vessel density showed no significant change over time. Next, these datasets were used for quantitative analysis by setting the tumor cell channel (GFP) at a threshold to count any clusters greater than 50 pixels2 (~0.0013mm2) and with greater than the mean + 2 standard deviations of the background signal while avoiding noise/artifacts from very small regions (<~0.001mm2). This allowed for computation of the total tumor area including average area of each cluster. In addition, the area moment, which describes the basic directional growth pattern of the tumor cells, was quantified by multiplying a cluster distance from the center of mass by its area. Conclusions: A key novel finding from structured illumination imaging data was the observation of LVI occurring early, similar with the clinical presentation in IBC patients. This model was able to effectively track tumor cluster migration. This approach of short-term longitudinal imaging time frame in studying transient or dynamic events of diffuse, collectively migrating tumor cells in the local environment and quantitative analysis of the tumor area, motility and vessel characteristics is an innovation that can be used to investigate other cancer cell types exhibiting LVI. Funding in part by DoD W81XWH-17-1-0297; W81XWH201053 (GRD); ACS Mission Boost grant MBG-20-141-01 (GRD) and NIH grant P30-CA014236 (Imaging Core/GMP) Citation Format: Gayathri R Devi, Dorababu Sannareddy, Alexandra Bennion, Ashlyn Rickard, Douglas C Rouse, Mark W Dewhirst, Gregory M Palmer. Monitoring Lymphovascular Invasion and Tumor Growth of Inflammatory Breast Cancer in a Murine Lymphatic Reporter Window Chamber Model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-06.

Proteomics analysis: inhibiting the expression of P62 protein by chloroquine combined with dacarbazine can reduce the malignant progression of uveal melanoma

BackgroundAlthough uveal melanoma (UM) at the early stage is controllable to some extent, it inevitably ultimately leads to death due to its metastasis. At present, the difficulty is that there is no way to effectively tackle the metastasis. It is hypothesized that these will be treated by target molecules, but the recognized target molecule has not yet been found. In this study, the target molecule was explored through proteomics.MethodsTransgenic enhanced green fluorescent protein (EGFP) inbred nude mice, which spontaneously display a tumor microenvironment (TME), were used as model animal carriers. The UM cell line 92.1 was inoculated into the brain ventricle stimulating metastatic growth of UM, and a graft re-cultured Next, the UM cell line 92.1-A was obtained through monoclonal amplification, and a differential proteomics database, between 92.1 and ectopic 92.1-A, was established. Finally, bioinformatics methodologies were adopted to optimize key regulatory proteins, and in vivo and in vitro functional verification and targeted drug screening were performed.ResultsCells and tissues displaying green fluorescence in animal models were determined as TME characteristics provided by hosts. The data of various biological phenotypes detected proved that 92.1-A were more malignant than 92.1. Besides this malignancy, the key protein p62 (SQSTM1), selected from 5267 quantifiable differential proteomics databases, was a multifunctional autophagy linker protein, and its expression could be suppressed by chloroquine and dacarbazine. Inhibition of p62 could reduce the malignancy degree of 92.1-A.ConclusionsAs the carriers of human UM orthotopic and ectopic xenotransplantation, transgenic EGFP inbred nude mice clearly display the characteristics of TME. In addition, the p62 protein optimized by the proteomics is the key protein that increases the malignancy of 92.1 cells, which therefore provides a basis for further exploration of target molecule therapy for refractory metastatic UM.

High Incidence of Lymph-node Metastasis in a Pancreatic-cancer Patient-derived Orthotopic Xenograft (PDOX) NOG-Mouse Model.

Our laboratory pioneered the patient-derived orthotopic xenograft (PDOX) model. An important goal of PDOX-model development is facile visualization of metastasis in live mice. In the present report we evaluated tumor growth and metastasis in pancreatic cancer PDOX NOG [Non-obese diabetes (NOD)/Scid/IL2Rγnull]-and nude-mouse models using red fluorescent protein (RFP)-expressing tumor stroma to visualize the primary tumor and metastasis. A patient-derived pancreatic cancer was initially implanted in transgenic RFP-expressing nude mice. Then, tumor fragments, which acquired RFP expressing stroma while growing in RFP-expressing nude mice were orthotopically implanted in nude and NOG mice. The primary pancreatic tumor and metastasis were observed 8 weeks after implantation. Lymph-node metastases expressing red fluorescence were detected only in NOG mice. Significantly faster growth of primary pancreatic tumors and a higher incidence of lymph-node metastasis occurred in NOG mice compared to nude mice. RFP-expressing tumor stroma, which traffics together with cancer cells to lymph nodes, is useful to observe tumor behavior, such as lymph-node metastasis in a PDOX NOG-mouse model which can be used for evaluation of novel anti-metastatic agents, as well as personalized therapy to identify effective drugs.

Color-Coded Imaging of Cancer and Stromal-Cell Interaction in the Pancreatic-Cancer Tumor Microenvironment (TME).

The tumor microenvironment (TME) contains stromal cells in a complex interaction with cancer cells. This relationship has become better understood with the use of fluorescent proteins for in vivo imaging, originally developed by our laboratories. Spectrally-distinct fluorescent proteins can be used for color-coded imaging of the complex interaction of the tumor microenvironment in the living state using cancer cells expressing a fluorescent protein of one color and host mice expressing another-color fluorescent protein. Cancer cells engineered in vitro to express a fluorescent protein were orthotopically implanted into transgenic mice expressing a fluorescent protein of a different color. Confocal microscopy was then used for color-coded imaging of the TME. Color-coded imaging of the TME has enabled us to discover that stromal cells are necessary for metastasis. Patient-derived orthotopic xenograft (PDOX) tumors were labeled by first passaging them orthotopically through transgenic nude mice expressing either green, red, or cyan fluorescent protein in order to label the stromal cells of the tumor (Yang et al., Cancer Res 64:8651-8656, 2004; Yang et al. J Cell Biochem 106: 279-284, 2009). The colored stromal cells become stably associated with the PDOX tumors through multiple passages in transgenic colored nude mice or non-colored nude mice. The fluorescent protein-expressing stromal cells included cancer-associated fibroblasts and tumor-associated macrophages. Color-coded imaging enabled the visualization of apparent fusion of cancer and stromal cells. Color-coded imaging is a powerful tool visualizing the interaction of cancer and stromal cells during cancer progression and treatment.

Color-Coded Imaging of the Tumor Microenvironment (TME) in Human Patient-Derived Orthotopic Xenograft (PDOX) Mouse Models.

The tumor microenvironment (TME) contains stromal cells in a complex interaction with cancer cells. This relationship has become better understood with the use of fluorescent proteins for in vivo imaging, originally developed by our laboratories. Spectrally distinct fluorescent proteins can be used for color-coded imaging of the complex interaction of the tumor microenvironment in the living state using cancer cells expressing a fluorescent protein of one color and host mice expressing another color fluorescent protein. Cancer cells engineered in vitro to express a fluorescent protein were orthotopically implanted into transgenic mice expressing a fluorescent protein of a different color. Confocal microscopy was then used for color-coded imaging of the TME. Color-coded imaging of the TME has enabled us to discover that stromal cells are necessary for metastasis. Patient-derived orthotopic xenograft (PDOX) tumors were labeled by first passaging them orthotopically through transgenic nude mice expressing either green, red, or cyan fluorescent protein in order to label the stromal cells of the tumor. The colored stromal cells become stably associated with the PDOX tumors through multiple passages in transgenic colored nude mice or noncolored nude mice. The fluorescent protein-expressing stromal cells included cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). Using this model, specific cancer cell or stromal cell targeting by potential therapeutics can be visualized. Color-coded imaging enabled the visualization of apparent fusion of cancer and stromal cells. Color-coded imaging is a powerful tool visualizing the interaction of cancer and stromal cells during cancer progression and treatment.

Restoration of Normal Blood Flow in Mouse Models of Sickle Cell Vaso-Occlusion Following Intravenous or Subcutaneous Administration of a Highly Potent E-Selectin Specific Inhibitor

Background: In sickle cell disease (SCD), recurrent episodes of vaso-occlusive crisis (VOC) contribute to morbidity and accelerated mortality. Analysis of the RESET Phase 3 study demonstrated that administration of the pan-selectin inhibitor, rivipansel, early in the course of VOC was associated with efficacy outcomes in favor of rivipansel, suggesting that reversal of vaso-occlusion may be achieved by treatment early in VOC. While both P- and L-selectin likely contribute to the underlying inflammatory cascade leading to VOC, previous studies have shown that E-selectin expression on the vascular luminal surface appears to be crucial for adhesion of sickled (SS)-RBC and leukocytes to the endothelium. GMI-1687 is an innovative potent E-selectin specific antagonist that demonstrates high bioavailability following subcutaneous (SC) administration. In the current investigation, we report on in vitro and in vivo studies evaluating the activity of GMI-1687 in SCD mouse models of VOC. Methods: Townes transgenic mice and nude mice transfused with human SS-RBC were used as in vivo models of SCD. VOC was initiated by TNF injection in both models. Adhesion to inflamed venules and development of VOC was recorded using intravital microscopy at sites of window chamber implants. The in vivo activity of E-selectin inhibition with GMI-1687 was assessed following IV injection in the nude mouse and Townes transgenic models, and SC injection in the nude mouse model of SCD. Results: In an initial study, IV treatment with saline or GMI-1687 was initiated 30 min post infusion of SS-RBC (t30) into TNF-treated mice, a time during which sickle cells are adherent to inflamed venules and VOC has initiated, and repeated 30 minutes later (t60). As expected, mice treated with saline alone showed marked adhesion of human SS-RBC to venules (61% occlusion of the total vessels recorded at t30) with evident blood stasis. Under these conditions, GMI-1687 at 40 μg/kg significantly reversed SS-RBC adhesion and VOC at both t30 and t60 (p=0.002 and 0.0073, respectively, compared to saline treatment). This led to restoration of blood flow in 85% of the venules in GMI-1687 treated mice vs. 18% of vessels with normal blood flow in the control saline treated group. Because of reduced sickle cell adhesion and VOC, the number of circulating SS-RBC increased by approximately 6-fold in mice treated with GMI-1687. Similar anti-adhesive and anti-VOC activities of GMI-1687 treatment were obtained in the Townes transgenic model of SCD where following IV administration of the E-selectin antagonist, adhesion of sickled RBCs was significantly diminished and as a result, the percentage of the total occluded vessel segments was reduced by 80-90%. We next assessed the activity of SC administration with 40 μg/kg GMI-1687 on human SS-RBC adhesion and development of VOC in the nude mouse model. Similar to the IV experiments, GMI-1687 initiated at t30 and t60 post SS-RBC infusion significantly reduced SS-RBC adhesion in inflamed vessels (69% reduction at t30, and 63% at t60, p=0.0001). The reduction in SS-RBC adhesion led to a lower incidence of VOC (2% compared to 30% of vessels in mice injected with saline), and a 5.1-fold improvement (p=0.0315) in the number of circulating human SS-RBC. It is noteworthy that the effective dose of GMI-1687 used in these IV and SC studies resulting in the restoration of normal blood flow was approximately 500-fold less than that reported for rivipansel. Conclusions: These data demonstrate that the administration of the potent and E-selectin specific compound GMI-1687 was effective in restoring blood flow in two mouse models of SCD, and support the development of GMI-1687 formulated for SC use and self-administration with the potential for early intervention of VOC. Disclosures Thamilarasan: GlycoMimetics: Research Funding. Fogler:GlycoMimetics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Zennadi:GlycoMimetics: Research Funding.

Abstract P1-03-03: Multichannel serial imaging of transgenic, preclinical murine models provides the first quantitative analysis of the unusual growth kinetics and lymph-vascular invasion of patient-derived inflammatory breast cancer cells and tumor emboli

Abstract Background: Lymphovascular invasion (LVI), a critical feature of advanced cancers, is a major route of metastatic dissemination. Of the clinically-distinct types of breast cancer, the most lethal variant is inflammatory breast cancer (IBC) where LVI is a histopathological hallmark. Majority of IBC patients lack a distinct, solid tumor and instead present with diffuse tumor cell clusters cells, also termed as tumor emboli, in the breast and dermal lymphatics, which contributes to the clinical breast-skin symptoms and postulated to provide an efficient path to metastatic spread. The steps in lymphatic dissemination are challenging to study in vitro, and in vivo models are limited. Our goal was to develop new in vivo models that would enable direct visualization and quantitation of local tumor cell growth and tumor-lymphatic vessel interactions for improved understanding of the unique IBC tumor biology and for drug discovery. Methods: mCherry/GFP-expressing non-IBC (4T1, E0771) and IBC cells derived from patient untreated primary tumors (SUM149/basal and SUM190/Her2+) were implanted into the fascia in the center of a window chamber in the skin-fold or mammary fat pad. In order to allow direct study of tumor cell–lymphatic vessel interaction, we generated transgenic nude mice with fluorescent lymphatics [tdTomato fluorophore under control of a Prox1 promoter, encodes a transcription factor (prospero-related homeobox 1) necessary for the formation and maintenance of lymphatic vessels]. Multichannel microscopy was employed to serially quantify tumor growth, tumor spread and lymph-tumor interactions. Using this model, we also optimized implantation and live imaging of pre-formed IBC-cell derived tumor emboli generated by simulating the lymphatic sheer stress in culture. Results: Compared to non-IBC cells, which formed solid tumors that increased in size over a 14 day period, IBC models exhibited a diffuse and disseminating phenotype, within 24h, rather than a centralized tumor mass, similar to the disease presentation in patients. Using a threshold analysis in ImageJ, we quantified tumor area (as a metric of tumor growth), the tumor motility, and the linear density of lymph and blood vessels. Briefly, we binarized each fluorescent channel image such that the tumor or lymph signal was positive; then, an algorithm drew regions of interest (ROIs) around each defined tumor-cell cluster. The ROIs were used to calculate the parameters describing tumor growth and motility (distance of the tumor from the center of mass (CM)), and the area moment (the movement of the clusters relative to the CM) at each timepoint. The linear vascular density was calculated from the distance of segments drawn through the center of large vessels for each mouse, which provided quantitative data describing tumor cell or IBC emboli and lymphovascular interactions, suggesting that there is a specific microenvironment necessary for the unique phenotype and dissemination pattern exhibited by IBC tumor cells. Conclusions: Despite the poor prognosis of IBC, the clinical implications of how and why tumor emboli form and how they survive to migrate into the lymphatics is not defined. These in vivo models provide in-depth imaging and quantitative measurements of locoregional invasion, tumor cell-lymphatic or endothelial vessel interactions of implanted tumor cells or tumor emboli. This model has the potential to be extended to study other cancer cell types exhibiting LVI as well as a screening metric for IBC therapies. Supported by DOD-Breakthrough-W81XWH-17-1-029, IBC Research Foundation, DCI pilot funds, IBC Network Foundation, Duke Surgery Gardner Award. Citation Format: Ashlyn Rickard, Pranalee Patel, Scott J Sauer, Mark W Dewhirst, Gregory M Palmer, Gayathri R Devi. Multichannel serial imaging of transgenic, preclinical murine models provides the first quantitative analysis of the unusual growth kinetics and lymph-vascular invasion of patient-derived inflammatory breast cancer cells and tumor emboli [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-03-03.

Peritoneal Metastases in a Patient-derived Orthotopic Xenograft (PDOX) Model of Colon Cancer Imaged Non-invasively via Red Fluorescent Protein Labeled Stromal Cells.

Patient-derived orthotopic xenograft (PDOX) models have patient-like clinical features and may be imaged, in case of some cancers, by passaging of the tumors through transgenic nude mice expressing red-fluorescent protein (RFP) where they stably acquire RFP expressing stroma. The aim of the present study was to quantify red fluorescent area and intensity in colon-cancer peritoneal metastases in PDOX models in non-transgenic nude mice after passage in RFP transgenic nude mice by non-invasive external fluorescence imaging. Tumor fragments originating from a colon cancer patient with peritoneal metastases were implanted in transgenic RFP nude mice. Resultant tumors were harvested, and fragments were implanted in the same strain a second time. Passaged tumors stably acquired RFP-expressing stroma from their transgenic hosts. The tumor with RFP-expressing stromal cells were harvested and implanted orthotopically in non-transgenic nude mice. At eight weeks post-implantation, non-invasive external RFP images were obtained. RFP area and intensity were measured and correlated with tumor weight and volume. Metastatic patient colon cancer can be stably and brightly labeled by passage in transgenic RFP-expressing nude mice such that tumor growth could be non-invasively imaged. Tumor growing could be non-invasively imaged when passaged to non-transgenic nude mice. A strong correlation between fluorescence intensity and area values with tumor weight and volume were established by external fluorescence imaging. This new tumor model of metastatic colon cancer can be used to evaluate novel therapeutics in real time for this recalcitrant disease.

Detection of Metastasis in a Patient-derived Orthotopic Xenograft (PDOX) Model of Undifferentiated Pleomorphic Sarcoma with Red Fluorescent Protein.

Undifferentiated pleomorphic sarcoma (UPS) is a common soft tissue sarcoma and highly recalcitrant. We have previously developed patient-derived orthotopic xenograft (PDOX) mouse models of UPS and other major sarcoma types. Unlike PDOX models of other cancer types, it has been difficult to demonstrate metastasis in the sarcoma PDOX models. To visualize metastasis at high resolution in the UPS PDOX model, established tumor fragments were implanted in transgenic nude mice expressing red fluorescent protein (RFP) for one passage. The tumors acquired RFP-expressing stroma from transgenic host. UPS tumor with RFP stromal cells were harvested and implanted orthotopically in non-transgenic nude mice. After six weeks of UPS tumor growth in the PDOX model, the primary tumor was imaged non-invasively and lung, liver, and spleen were resected and imaged ex-vivo in order to visualize the presence of RFP, with a FluorVivo® imaging system and FV1000® confocal laser microscope, respectively. The primary tumor was imaged non-invasively. Confocal microscopy visualized the presence of RFP in the lung and liver indicating metastases in these organs. This is the first report of metastasis in a sarcoma PDOX model. This study should prove very useful to screen for anti-metastatic drugs for the PDOX donor patients and to understand the metastatic process in sarcoma.

Relevant molecular characteristics analysis on malignant transformation of interstitial cells induced by tumor stem cells in glioma microenvironment

Objective: A variety of interstitial cells in tumor microenvironment (TME) based on glioma stem cells(GSC) have the function to promote malignant progression of tumors, but whether these interstitial cells have already undergone malignant transformation and their related molecular characteristics are still poorly understood. Methods: Human SU3-RFP glioma stem cells (GSC) stably transfected with red fluorescent protein (RFP) and interstitial cells from enhanced green fluorescent protein (EGFP) transgenic nude mice were co-cultured in vitro. SU3-RFP cells were also inoculated in different tissues of EGFP-Balb/c nude mice. Immortal EGFP(+) cells were monocloned either from co-culture cells in vitro, or from their xenografts in vivo. These immortal EGFP(+) cells were confirmed to bear characteristics of tumor cell via chromosomal analysis and tumorigenicity assay. Related molecular phenotypes of these cells were further detected through RT-PCR, flow cytometry and immunochemistry(IHC) techniques. Results: (1) Two EGFP(+) cell lines were obtained in vitro, and 5 EGFP(+) cell lines were obtained in vivo tumorigenic experiments. Seven EGFP(+) cell lines all have characteristics of self-renewal, heteroploid of chromosomes and 100% tumorigenicity. (2) Cell surface marker analysis showed cell origin of these cell lines were macrophages (tMΦ1 and tMΦ2 ), dendritic cells (tDC1 and tDC2), fibroblasts (tFB), oligodendrocytes (tOG) and BMSC cells (tBMSC), respectively. (3)All of these seven cell lines co-expressed Sca-1 and c-myc, and have Sox-2 or Nanog expression also, which suggest that they may bear molecular characteristics of mesenchymal stem cells or pluripotent stem cells. Conclusions: (1) Tumor stromal cells in TME have undergone malignant transformation, which is related to the tissue remodeling of TME by GSCs, and not limit to the specific type of their parasitic tissues. (2) Tumor cells originated from GSC and tumor interstitial cells, respectively, are two major types of tumor cells with different origins in glioma parenchyma, can not be simply regarded as tumor heterogeneity, transformed interstitial cells of TME may have the potential to serve as new targets for target diagnosis and therapy.

Imaging the interaction of α v integrin‐GFP in osteosarcoma cells with RFP‐expressing host stromal cells and tumor‐scaffold collagen in the primary and metastatic tumor microenvironment

Human osteosarcoma 143B cells were previously stably transfected with an αv integrin green flourescent protein (GFP) vector. 143B cells expressing αv integrin-GFP were transplanted orthotopically in the tibia of transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). The primary tumors acquired RFP-expressing stroma and were passaged orthotopically in the tibia in noncolored nude mice, which maintained the RFP stroma. The interaction of αv integrin-GFP expression in 143B cells with RFP-expressing host stromal cells was observed by confocal microscopy using the Olympus FV1000. Collagen fibers were imaged simultaneously in reflectance mode. The RFP-expressing stroma included cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) which persisted even 3 weeks after passage to nontransgenic nude mice. CAFs expressing RFP were aligned between collagen fibers and cancer cells expressing αv integrin-GFP. Six weeks after transplantation, pulmonary metastases expressing αv integrin-GFP could be identified. TAMs expressing RFP accompanied metastasized osteosarcoma cells expressing αv integrin-GFP in the lung. The current study demonstrates the importance of αv integrin interaction with stromal elements in osteosarcoma.

Visualizing the Tumor Microenvironment by Color-coded Imaging in Orthotopic Mouse Models of Cancer.

The tumor microenvironment (TME) contains stromal cells in a complex interaction with cancer cells. This relationship has become better understood with the use of fluorescent proteins for in vivo imaging, originally developed by our laboratories. Spectrally-distinct fluorescent proteins can used for color-coded imaging of the complex interaction of the tumor microenvironment in the living state using cancer cells expressing a fluorescent protein of one color and host mice expressing another-color fluorescent protein. Cancer cells engineered in vitro to express a fluorescent protein were orthotopically implanted into transgenic mice expressing a fluorescent protein of a different color. Confocal microscopy was then used for color-coded imaging of the TME. Color-coded imaging of the TME has enabled us to discover that stromal cells are necessary for metastasis. Patient-derived orthotopic xenograft (PDOX) tumors were labeled by first passaging them orthotopically through transgenic nude mice expressing either green, red, or cyan fluorescent protein in order to label the stromal cells of the tumor. The colored stromal cells become stably associated with the PDOX tumors through multiple passages in transgenic colored mice or non-colored mice. The fluorescent protein-expressing stromal cells included cancer-associated fibroblasts and tumor-associated macrophages. The cancer cells in PDOX models can also be labeled with a telomerase-dependent adenovirus containing the gene for green fluorescent protein. Using this model, specific cancer-cell or stromal-cell targeting by potential therapeutics can be visualized. Color-coded imaging enabled the visualization of apparent fusion of cancer and stromal cells. Color-coded imaging is a powerful tool visualizing the interaction of cancer and stromal cells during cancer progression and treatment.

Experimental study of glioma stem cell-mediated immune tolerance in tumor microenvironment

Objective: To investigate the tumor microenvironment of immune tolerance induced by glioma stem cells (GSC). Methods: Human GSC SU3 cells transfected with red fluorescent protein (SU3-RFP) gene were implanted into the brain, subcutis (armpit and foot), liver and abdominal cavity of transgenic green fluorescence protein (GFP) nude mice to establish RFP(+) /GFP(+) dual fluorescence solid tumor model. The re-cultured cells derived from implanted tumor tissues, SU3-RFP cells co-cultured with peritoneal fluid of transgenic GFP nude mice and malignant ascites of tumor-bearing mice were observed by fluorescence microscopy and real-time video image tracing to analyze the microenvironment of immune tolerance mediated by RFP(+) /GFP(+) implanted tumor. Results: Dual fluorescence labeled frozen section showed that all of cells in the tumor microenvironment were GFP(+) , while the pressed tissue-patch showed that the tumor blood vessels exhibited a RFP(+) /GFP(+) double-positioning yellow. In the GFP single fluorescence labeled tumor tissue, all of cells in the microenvironment were green, including tumor edge, necrotic foci and blood vessel. Among them, CD68(+) , F4/80(+) , CD11c(+) , CD11b(+) and CD80(+) cells were observed. In the dual fluorescence labeled co-cultured cells, the phagocytosis and fusion between green host cells and red tumor cells were also observed, and these fusion cells might transfer to the malignant dendritic cells and macrophages. Conclusions: The tumor microenvironment of immune tolerance induced by GSC is not affected by the tissue types of tumor-inoculated sites, and the immune tolerance mediated by inflammatory cells is associated with the inducible malignant transformation, which may be driven by cell fusion.

Systemic administration of β-glucan of 200 kDa modulates melanoma microenvironment and suppresses metastatic cancer

ABSTRACTConverting an immunosuppressive melanoma microenvironment into one that favors the induction of antitumor immunity is indispensable for effective cancer immunotherapy. In the current study we demonstrate that oat-derived β-(1-3)-(1-4)-glucan of 200 kDa molecular size (BG34-200) previously shown to mediate direct interaction with macrophages could alter the immune signature within melanoma microenvironment. Systemic administration of BG34-200 resulted in reversion of tolerant melanoma microenvironment to an immunogenic one that allows M1-type activation of macrophages, the induction of pro-inflammatory cytokines/chemokines including IFN-γ, TNF-α, CXCL9, and CXCL10, and enhanced IRF1 and PD-L1 expression. In turn, BG34-200 induced a superior antitumor response against primary and lung metastatic B16F10 melanoma compared to untreated controls. The enhanced tumor destruction was accompanied with significantly increased tumor infiltration of CD4+ and CD8+ T cells as well as elevated IFN-γ in the tumor sites. Systemic administration of BG34-200 also provoked systemic activation of tumor draining lymph node T cells that recognize antigens naturally expressing in melanoma (gp100/PMEL). Mechanistic studies using CD11b-knockout (KO), CD11 c-DTR transgenic mice and nude mice revealed that macrophages, DCs, T cells and NK cells were all required for the BG34-200-induced therapeutic benefit. Studies using IFN-γ-KO transgenic mice showed that IFN-γ was essential for the BG34-200-elicited antitumor response. Beyond melanoma, the therapeutic efficacy of BG34-200 and its immune stimulating activity were demonstrated in a mouse model of osteosarcoma. Together, BG34-200 is highly effective in modulating antitumor immunity. Our data support the potential therapeutic use of this novel immune modulator in the treatment of metastatic melanoma.

Abstract 2862: Tumor-targeting salmonella typhimurium a1-r inhibited angiogenesis of osteosarcoma cells visualized by color-coded imaging in the in vivo absorbable gelatin sponge assay

Abstract INTRODUCTION We already have reported a color-coded imaging model that showed the sponge agngiogenesis assay using absorbable gelatin sponge implanted in nestin-driven green fluorescent protein (ND-GFP) nude mice and that osteosarcoma cells promote angiogenesis in this sponge assay. We report here that Salmonella typhimurium A1-R (A1-R) inhibits angiogenesis of osteosarcoma cells in the sponge angiogenesis assay in ND-GFP mice. METHODS Sponge was initially transplanted subcutaneously in the flank of transgenic ND-GFP nude mice. Seven days after transplantation of sponge, skin flaps were made and 143B-RFP human osteosarcoma cells expressing red fluorescent protein (RFP) were injected into the transplanted sponge. After establishment of tumors in the sponge, the control-group mice were treated with PBS via tail-vein injection and A1-R treated group with A1-R likewise. Skin flaps were made at days 14, 21, and 28 after transplantation of the sponge to allow imaging of vascularization in the sponge using a variable-magnification small animal imaging system and confocal fluorescence microscopy. RESULTS: Vessel length in the sponge was measured after treatment with FV10-ASW Fluoview software. Nascent blood vessels grew in the sponge in a time-dependent manner. A random 3 fields were quantified in each group). The mean length of ND-GFP expressing blood vessels in the sponge in mice with A1-R treated group were 9.40, 12.40, and 10.12 mm/mm2, at days 14, 21, and 28, respectively. The mean length of ND-GFP expressing blood vessels in the sponge in mice of the control group were 8.86, 14.50, and 15.81 mm/mm2 on day 14, 21, and 28, respectively. ND-GFP expressing blood vessels of the mice in the osteosarcoma cells treated with A1-R had shorter vessels than the control group (on days 28, P < 0.05), suggested the extent of nascent blood vessel growth was significantly inhibited by A1-R treatment. Besides, we evaluated the RFP colored area of the sponge in both groups, which almost represent the viable tumor size. The mean RFP colored area of A1-R treated group in the sponge were 4.49, 7.53 and 8.15 mm2, at days 14, 21, and 28, respectively and that of control group were 3.78, 6.40, and 7.49 mm2 at days 14, 21, and 28, respectively. The size of possibly viable RFP colored tumor area between A1-R treated group and control group at days 28 did not show significant area difference, indicated A1-R treated osteosarcoma cells showed shorter nascent vessel length than control group, not due to the tumor size difference but due to the difference of angiogenesis inhibitory effect. As a result of both outcomes above, the extent of nascent blood vessel growth was significantly inhibited by A1-R treatment on days 28. CONCLUSION: In the present study, we reported the effect of angiogenesis inhibition of A1-R in vivo sponge assay, and suggest A1-R has potential for anti-angiogenic target therapy for osteosarcoma. Citation Format: Tasuku Kiyuna. Tumor-targeting salmonella typhimurium a1-r inhibited angiogenesis of osteosarcoma cells visualized by color-coded imaging in the in vivo absorbable gelatin sponge assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2862. doi:10.1158/1538-7445.AM2017-2862

Abstract 2874: Inhibition of tumor angiogenesis by Salmonella typhimurium A1-R in the in vivo Gelfoam color-coded imaging assay

Abstract We previously demonstrated a color-coded angiogenesis imaging model using Gelfoam® implanted in nestin-driven green fluorescent protein (ND-GFP) nude mice in which nascent blood vessels express GFP. In this assay, we demonstrated osteosarcoma promotes angiogenesis. We report here that Salmonella typhimurium A1-R (S. typhimurium A1-R) inhibits angiogenesis of osteosarcoma cells in the Gelfoam® angiogenesis assay in ND-GFP mice. Gelfoam® was transplanted subcutaneously in the flank of transgenic ND-GFP nude mice. Seven days after transplantation, skin flaps were made and 143B human osteosarcoma cells, expressing red fluorescent protein (RFP), were injected in the transplanted Gelfoam®. After establishment of tumor in the Gelfoam®, the control group mice were treated with PBS by tail vein injection. The treated group was treated by S. typhimurium A1-R, also by tail vein injection. Skin flaps were made at days 14, 21, and 28 after transplantation of the Gelfoam® in order to allow imaging of vascularization. The OV100 variable magnification small animal imaging system and FV1000 confocal fluorescence microscope were used to visualize vascularization. Nascent blood vessels grew in the Gelfoam® in a time-dependent manner. A random 3 fields were quantified in each group. The mean length of ND-GFP-expressing blood vessels in the Gelfoam® in mice were treated with S. typhimurium A1-R was 9.40, 12.40, and 10.12 mm/mm2, at days 14, 21, and 28, respectively. The mean total length of ND-GFP-expressing blood vessels in the Gelfoam® in mice of the control group were 8.86, 14.50, and 15.81 mm/mm2 on day 14, 21, and 28, respectively. ND-GFP expressing blood vessels of the mice in the osteosarcoma cells treated with S. typhimurium A1-R had shorter vessels than the control group (on day-28, P<0.05). There was not a significant difference in tumor size between the 2 groups, thereby indicating a specific inhibition of angiogenesis by S. typhimurium A1-R. In the present study, we reported that S. typhimurium A1-R inhibited angiogenesis of human osteosarcoma cells in the in vivo Gelfoam® color-coded imaging assay. Citation Format: Tasuku Kiyuna, Yasunori Tome, Takashi Murakami, Ming Zhao, Fuminori Kanaya, Robert M. Hoffman. Inhibition of tumor angiogenesis by Salmonella typhimurium A1-R in the in vivo Gelfoam color-coded imaging assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2874. doi:10.1158/1538-7445.AM2017-2874

Analysis of Stroma Labeling During Multiple Passage of a Sarcoma Imageable Patient-Derived Orthotopic Xenograft (iPDOX) in Red Fluorescent Protein Transgenic Nude Mice.

A patient-derived orthotopic xenograft (PDOX) model of undifferentiated pleomorphic sarcoma (UPS) was previously established that acquired red fluorescent protein (RFP)-expressing stroma by growth in an RFP transgenic nude mouse. In the present study, an imageable PDOX model (iPDOX) of UPS was established by orthotopic implantation in the biceps femoris of transgenic RFP nude mice. After the tumors grew to a diameter of 10 mm, they were harvested and the brightest portion of the tumors were subsequently orthotopically transplanted to both RFP and non-colored nude mice. The UPS PDOX tumor was again transplanted to RFP transgenic and non-colored nude mice, and finally a 3rd passage was made in the same manner. Five UPS tumors from each passage in both RFP and non-colored mouse models were harvested. The FV1,000 confocal microscope was used to visualize and quantitate the RFP area of the resected tumors. The average percent fluorescent area in the first passage of RFP mice was 34 ± 22%; in the second passage, 34 ± 20%; and 36 ± 11% in the third passage of RFP transgenic nude mice. The average tumor RFP area in the first passage from RFP mice to non-colored mice was 20 ± 7%; in the second passage, 28 ± 11%; in the third passage was 27 ± 13%. The present results demonstrate the extensive and stable acquisition of stroma by the UPS-tumor growing orthotopically in transgenic RFP nude mice (iPDOX). This model can be used for screening for effective drugs for individual patients and drug discovery. J. Cell. Biochem. 118: 3367-3371, 2017. © 2017 Wiley Periodicals, Inc.

Hyperbaric oxygen therapy sensitizes nimustine treatment for glioma in mice.

Nimustine (ACNU) has antitumor activities in patients with malignant glioma. Hyperbaric oxygen (HBO) may enhance the efficacy of certain therapies that are hampered by the hypoxic microenvironment. We examined the combined effects of ACNU and HBO in a GFP transgenic nude mice bearing human glioma model. Mice inoculated with human glioma cells SU3 were randomly divided into the four groups: (A) the control group, (B) the HBOT (HBO therapy) group, (C) the ACNU group, and (D) the HBOT+ACNU group. Tumor size was measured at the indicated time intervals with a caliper; mice were sacrificed 28 days after treatment, and immunohistochemistry staining and western blot analysis were carried out. By the end of the trial, the tumor weights of groups A, B, C, and D were (P < 0.05), 6.03 ± 1.47, 4.13 ± 1.82 (P < 0.05), 2.39 ± 0.25 (P < 0.05), and 1.43 ± 0.38 (P < 0.01), respectively. The expressions of TNF‐α, MMP9, HIF‐α, VEGF, NF‐κB, and IL‐1β were associated with the infiltration of inflammatory cells and the inhibition rate of tumor cells. Hyperbaric oxygen therapy (HBOT) could inhibit glioma cell proliferation and inflammatory cell infiltration, and exert a sensitizing effect on ACNU therapy partially through enhancing oxygen pressure (PO2) in tumor tissues and lower expression levels of HIF‐1α, TNF‐α, IL‐1β, VEGF, MMP9, and NF‐κB.

Abstract 4221: Imaging formation of the tumor microenvironment in orthotopic liver tumors in red fluorescent protein (RFP) transgenic nude mice

Abstract We report here dual-color imaging of the formation of the TME in orthotopic liver cancer in transgenic red fluorescent protein (RFP) nude mice, which express RFP in all organs. Non-colored HUH-7 human hepatoma cells were injected in the spleen of RFP nude mice to establish an orthotopic liver cancer model. TME formation in the liver tumor was observed using the Olympus OV100 small animal fluorescence imaging system. Non-colored liver cancer cells formed colonies in the liver 28 days after cell transplantation to the spleen. In the liver, RFP host-mouse cells accumulated around and in the liver tumor as visualized by fluorescence imaging. A desmin and sirus-red positive area increased around and within the liver tumor over time. These results indicate cancer-associated fibroblasts (CAFs) were recruited by the liver metastasis suggesting that CAFs, along with the angiogenic tumor blood vessels, could serve as visible therapeutic targets. Citation Format: Atsushi Suetsugu, Yukihiko Hiroshima, Takuro Matsumoto, Kosuke Hasegawa, Miki Nakamura, Masahito Shimizu, Shigetoyo Saji, Hisataka Moriwaki, Michael Bouvet, Robert M. Hoffman. Imaging formation of the tumor microenvironment in orthotopic liver tumors in red fluorescent protein (RFP) transgenic nude mice. [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 4221.