Severe Combined Immunodeficiency Disease Mice Research Articles

Abstract 2835: Comparison of xenograft and syngeneic cancer cachexia models for preclinical drug testing

Abstract Cancer cachexia is a wasting syndrome characterized primarily by loss of skeletal muscle and adipose tissue mass and contributes greatly to overall cancer-related mortality (for recent review see Argiles 2023). An estimated 25-75% of cancer patients develop cachexia which leads to impacts on therapeutic tolerability, well-being, and eventually overall survival (Bossi 2021). Some preclinical mouse and rat cancer models manifest cachexia as they progress. In this study we established a syngeneic (immune competent) and a xenograft (immune incompetent) tumor model of cachexia with the intention of comparing the models with respect to typical study endpoints and to provide a platform for testing novel therapeutic agents aimed at inhibiting or reversing the effects of cachexia. The Colon26 syngeneic mouse colon adenocarcinoma model was reported as an experimental cancer cachexia model by Tanaka, et. al. in 1990. The model was developed in male BALB/c × DBA/2 F1 (first generation cross) mice. We typically implant Colon26 in female BALB/c mice for preclinical drug testing of anticancer agents and noticed a strong tendency for mice bearing Colon26 tumors to develop signs of cachexia on study, but not always consistently. A review of the literature showed there are clear gender differences with male cancer patients having higher prevalence of cachexia and greater muscle loss compared with female patients (Zhong 2020). Based on this we implanted Colon26 tumors in male BALB/c mice to establish the cachexia model. The HT1080 human fibrosarcoma model HT1080 was characterized as a cachexic model by Bernardo, et. al. in female severe combined immunodeficiency disease (SCID) mice. We developed the HT1080 model in male SCID mice based on the literature and previous experience. Both models were characterized by multiple endpoints including overall body weight loss, skeletal muscle weight loss, reduction of adipose tissue, and cardiac muscle loss, as well as tracking a broad panel of relevant cytokine levels at study endpoint. Both models generate robust wasting of skeletal muscle and adipose tissue providing an opportunity to assess novel anti-cachexia therapies. Citation Format: Kyle Draheim, Diana Gietl, Karyn Shinn, William Durham, Fei Zhao, Caitlin Thompson, Alexis Baldwin, Glenn Harris, Chassidy Hall, Patrick Fadden, Anya Avrutskaya. Comparison of xenograft and syngeneic cancer cachexia models for preclinical drug testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2835.

Effect of Cell Density of a Methacrylic Acid-Based Hydrogel Implant on Embedded Islet Function and Viability.

Subcutaneous delivery of islets in a methacrylic acid-based hydrogel may offer a functional cure for type 1 diabetes. Here we show in mice that the hydrogel is able to provide sufficient vasculature to support islet function and viability, when islets are used at a low islet volume fraction (i.e., cell density). The Krogh cylinder model was used to mathematically estimate the effect of implant volume, for a fixed islet dose (600 islet equivalents [IEQ]), on the minimum vessel density required to maintain sufficient pO2 within the graft. Modeling suggested that 200 μL implants would have low enough islet densities and enough vessels to have islets remain viable, but that 50 μL implants would not; this was confirmed experimentally through measurement of glucose level in streptozotocin-induced diabetic severe combined immunodeficiency disease (SCID/bg) mice, comparing 200 and 50 μL implants, both with 600 IEQ. Vessel densities were ∼20-30 vessels/mm2 independent of implant volume and vessels were sufficient to increase subcutaneous oxygen tension, as measured with microcapsules containing oxygen sensitive material (a platinum [Pt] porphyrin); both these results were determined without cells. These results are useful in thinking about the scale-up of this system to humans: to maintain a low islet density (∼0.5%), many more islets will require attention to the subcutaneous implant configuration to satisfy the oxygen needs of the cells.

Development of a HPLC-MS/MS Method to Assess the Pharmacokinetics and Tumour Distribution of the Dimethylarginine Dimethylaminohydrolase 1 Inhibitors ZST316 and L-257 in a Xenograft Model of Triple-Negative Breast Cancer in Mice.

We describe the development and validation of an HPLC-MS/MS method to assess the pharmacokinetics and tumour distribution of ZST316, an arginine analogue with inhibitory activity towards dimethylarginine dimethylaminohydrolase 1 (DDAH1) and vasculogenic mimicry, and its active metabolite L-257 in a xenograft model of triple-negative breast cancer (TNBC). The method proved to be reproducible, precise, and highly accurate for the measurement of both compounds in plasma and tumour tissue following acute and chronic (five days) intraperitoneal administration of ZST316 (30 mg/Kg daily) in six-week-old severe combined immunodeficiency disease (SCID) mice inoculated with MDA-MB-231 TNBC cells. ZST316 was detected in tumour tissue and plasma after 1 h (6.47 and 9.01 μM, respectively) and 24 h (0.13 and 0.16 μM, respectively) following acute administration, without accumulation during chronic treatment. Similarly, the metabolite L-257 was found in tumour tissue and plasma after 1 h (15.06 and 8.72 μM, respectively) and 24 h (0.17 and 0.17 μM, respectively) following acute administration of ZST316, without accumulation during chronic treatment. The half-life after acute and chronic treatment ranged between 4.4-7.1 h (plasma) and 4.5-5.0 h (tumour) for ZST316, and 4.2-5.3 h (plasma) and 3.6-4.9 h (tumour) for L-257. The results of our study demonstrate the (a) capacity to accurately measure ZST316 and L-257 concentrations in plasma and tumour tissue in mice using the newly developed HPLC-MS/MS method, (b) rapid conversion of ZST316 into L-257, (c) good intra-tumour penetration of both compounds, and (d) lack of accumulation of both ZST316 and L-257 in plasma and tumour tissue during chronic administration. Compared to a previous method developed by our group to investigate ZST316 in plasma, the main advantages of the new method include a wider range of linearity which reduces the need for dilutions and the combined assessment of ZST316 and L-257 in plasma and tumour tissue which limits the required amount of matrix. The new HPLC-MS/MS method is useful to investigate the in vivo effects of ZST316 and L-257 on vasculogenic mimicry, tumour mass, and metastatic burden in xenograft models of TNBC.

Abstract 132: The molecular basis of an evolutionary arms race between cancer cells and their host

Abstract Genetic and nongenetic changes can drive abnormal characteristics of cancer cells, including resistance to tumor growth. Through selective breeding of animals with the slowest growing tumor at 11 days following implantation over ten generations, we evolved resistance to tumor growth in two contrasting strains of mice: severe combined immunodeficiency disease (SCID) and immuno-competent C57BL/6. We found that the two strains evolved different strategies of resistance to the cancer. In the immunocompromised mice, skin fibroblasts infiltrated into and around the tumor to impede growth, while an enhanced immune response to tumor antigens, slowed tumor growth in the immunocompetent mice. However, when the experiment was extended beyond 11 days, tumor growth rapidly increased so that, at 28 days, it was comparable in size to the control animals. We investigated the molecular-level mechanisms underlying these resistance traits in SCID mice. We identified transcriptomic changes using RNA-Seq in both skin and tumor samples to compare the evolved mice (EV) with the original strain (WT) at 11 days and 28 days. We used DESeq2 and two-way ANOVAs on the normalized data to identify significant differences in gene expression. We adjusted p-values for multiple testing with the Benjamini-Hochberg false discovery correction and used a q value of less than 0.1 (and/or a greater than two-fold change) to determine the significance for differential expression levels. Comparing transcriptomic data from skin fibroblasts from evolved (EV) and non-evolved (WT) SCID mice at day 11 following implantation (i.e., the point of maximal tumor suppression) revealed 98 differentially expressed genes (DEGs). EV SCID fibroblasts showed increased expression of MicroRNAs, including MiR-7 and Mir5125, and acid metabolic pathways, such as Carbonic anhydrase III. A comparison of the tumor cells from day 11 and day 28 demonstrated substantial epigenetic evolution with 1353 DEGs. The tumor cells at day 28 adapted to the host response by increasing expression of genes for extracellular matrix (ECM) remodeling, mainly the metalloproteases gene family. Here we used selective breeding to allow evolution to reveal potential host strategies to suppress tumor growth. However, we find that, over time, tumor cells can evolve successful counter strategies to overcome host resistance. Our results provide novel insights into key dynamics of somatic evolution during carcinogenesis and highlight the critical roles of epigenetic modification and inheritance in these somatic eco-evolutionary dynamics. Citation Format: Arig A. Ibrahim-Hashim, Robert A. Gatenby, Christina L. Richards, Joel S. Brown. The molecular basis of an evolutionary arms race between cancer cells and their host [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 132.

Abstract 5932: Clonal mapping phylogenetic and transcriptomic analysis of an M36 PDX amputation lung metastatic osteosarcoma model

Abstract Introduction: Osteosarcoma is a primary malignant bone tumor characterized by the production of spindle cells resulting in immature bone formation. It has been revealed that metastatic cancers including osteosarcoma have a subset of cells with different phenotypic and genetic signatures conferring advantage to drive progression and drug resistance within the intra-tumorally heterogeneous population. However, the lack of a defined in vivo model that can identify the rare genetic subpopulation and recapitulate clonal evolution has been a challenge. In this study, we used an established model to track and identify the clonal subpopulations and assess the genetic signatures that confer specific advantages selecting for uncontrolled growth, adaptation and colonization in the lungs. Methods: We injected barcoded M36 PDX osteosarcoma cells intratibially in ten severe combined immunodeficiency disease (SCID) mice to track the clonal population/subpopulation of cells that metastasize the established primary tumors to the lungs. Limb amputation was performed on the tumor bearing leg upon detection of palpable tumor. Lung tissue was harvested post-amputation following death from metastasis or euthanasia. DNA and RNA from frozen tissues was extracted and analyzed by PCR, NGS, WES and RNA sequencing to map clonality, mutational and evolutionary profiles. Results: Limb amputation was performed in 7 mice with all mice surviving the surgery. The mean mouse survival following amputation was 46.9 days and the longest survival was 167 days. Four (40%) of the primary tumor bearing mice had lung metastasis. A Shannon-Wiener and Jaccard similarity index show a diversity in clonal architecture between the primary tumors of all mice injected on the same day and a diversity in clonal population in both the primary tumors and their matched lung metastatic nodules. Clonal mapping further revealed 21 unique clones shared in all primary tumors examined. However, the clonal frequencies and distribution varied within each primary tumor and across the tumor samples. Interestingly, clonal frequencies increased in the lung metastatic nodules when compared to their matched primary tumors. Four distinct clones that were present in all the primary tumors were also found in all lung metastatic samples. These unique clones, here in called “seeding clones” may play an important role in the lung metastatic process. Conclusions: We previously showed that the barcoded osteosarcoma PDX models are inter and intratumorally heterogenous both in vitro and in vivo. In the current model, we identify the multiple tumorigenic seeding clones that potentially act as drivers in the lung metastatic process. Furthermore, we show the dynamic evolutionary relationships between the clones by reconstructing a phylogenetic map of the M36 PDX tumor model. Funding: Swim Across America, Foster Foundation and the Barbara Epstein Foundation. Citation Format: Sylvester Jusu, Zhongting Zhang, Zhang Wendong, Zhaohui Xu, Michael Roth, Jonathan Gill, Richard Gorlick. Clonal mapping phylogenetic and transcriptomic analysis of an M36 PDX amputation lung metastatic osteosarcoma model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5932.

Abstract LB166: Evaluation of CAR-T anti-tumor response in a novel immunocompromised NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl mouse model

Abstract Background: The development of immunocompromised mice revolutionized the field of cancer and stem cell research. Due to the lack of mature B and T lymphocytes, SCID (severe combined immunodeficiency disease) mice are ideal for xenoengraftment of human cells and tissue, including patient-derived tumor cells, hematopoietic stem cells and T cells. Over the years, the SCID models have been implemented to increase the efficiency of engraftment and NSG (NOD-SCID;IL2Rgammanull) has become the most reliable in vivo human cancer model. Here we compared NSG with the novel mouse strain NCG for immune-oncology applications, in particular for CAR-T therapy. NCG (NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl) is a recently developed triple immunodeficient mouse strain, created by sequential CRISPR/Cas9 editing of the PRKDC and IL2rg loci in the NOD/Nju strain. For this purpose, we engrafted NALM-6 and CHLA-55 tumor cell lines and treated them with CD19 and GD2.CAR T cells, respectively. Results: Four different operators generally considered NCG mice to be easier to handle. NCG mice were a bit smaller and have a much smaller spleen compared to age-matched NSG mice. Tumor engraftment was particularly rapid in NCG while the dissemination to organs was comparable. Generally, NCG showed signs of sickness earlier and upon lower tumor burden. When treated with CAR-T cells, in vivo anti-tumor response was comparable, both in a leukemia mouse model (96.6% NSG vs 97.7% NCG) and in a disseminated neuroblastoma model (67.3% NSG vs 57.8% NCG).Conclusion: Our preliminary data show that NCG mice are a suitable immunocompromised host for CAR-T pre-clinical studies. Citation Format: Rashmi Bankoti, Amit Joshi, Lasse Jorgensen, Andreas Kongsgaard, Maddalena Adorno, Benedetta Nicolis di Robilant. Evaluation of CAR-T anti-tumor response in a novel immunocompromised NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB166.

A severe combined immunodeficiency disease mouse model of human adenocarcinoma with lepidic-predominant growth

Severe combined immunodeficiency disease (SCID) mice with human lepidic adenocarcinoma were established by the intrabronchial implantation of fresh surgically resected specimens. Human pulmonary adenocarcinoma tissue from 16 different cases was transplanted into SCID mice, and SCID mouse tumors were established from four of these cases (25%). Among the four tumors, the tumor cells of two SCID mice showed replacement lepidic growth of mouse alveolar structures accompanied by multiple intrapulmonary lesions. Human lung carcinoma cell lines showing lepidic growth are rare and the xenograft models using the SCID mouse model developed in the current study will be useful for analyzing the growth and/or progression patterns and clinical behavior of lepidic adenocarcinoma, the major histological subtype of human carcinoma of the lung.

Anti-tumor effects of 13-cis-retinoic acid combined with interferon α-2b in animal model of mantle cell lymphoma

目的评估13-顺式维甲酸（13cRA）和IFN-α-2b单用，以及二者联合应用对套细胞淋巴瘤（MCL）动物模型的抗肿瘤效应，并探讨其作用机制。方法构建MCL细胞株Jeko-1细胞重症联合免疫缺陷小鼠模型，将荷瘤小鼠随机分成阴性对照组（溶剂），高（200 mg/kg）、中（100 mg/kg）、低（50 mg/kg）13cRA剂量组，IFN-α-2b组，不同剂量13cRA联合IFN-α-2b组，阳性对照组（硼替佐米＋利妥昔单抗＋环磷酰胺），同时进行干预治疗。定期观察荷瘤小鼠肿瘤体积变化，计算相对肿瘤增殖率、抑瘤率。采用免疫组化法检测Ki-67的表达。采用缺口末端标记法检测肿瘤组织细胞凋亡情况。采用Western blot法检测Cyclin D1、caspase-9及视网膜神经胶质瘤蛋白（Rb）等的表达水平。结果①中、高剂量13cRA组及中、高剂量13cRA联合IFN-α-2b组的相对肿瘤增殖率分别为30%、37%、32％和33%。②低、中、高剂量13cRA组或其联合IFN-α-2b组的抑瘤率均较阴性对照组明显增高（P<0.05），不同剂量13cRA组间、单用IFN-α-2b组抑瘤率与阴性对照组比较差异均无统计学意义（P值均>0.05）。中剂量13cRA组或其联合IFN-α-2b组抑瘤率最高，分别为59.2%、62.6%，与阳性对照组（69.4%）差异无统计学意义（P>0.05）。③Ki-67在各组的表达差异无统计学意义（P=0.342）。④不同剂量13cRA组及其联合IFN-α-2b组凋亡细胞数均较阴性对照组明显增加（P<0.05），与阳性对照组差异无统计学意义（P=0.170）；阴性对照组凋亡细胞数与IFN-α-2b组差异无统计学意义（P=0.098）。⑤不同剂量13cRA联合IFN-α-2b组与阴性对照组比较，cycling D1及procaspase-9降低，cleaved caspase-9升高，与阳性对照组表达相当；不同剂量13cRA组与阴性对照组比较，则未见明显差异。结论在MCL动物模型中IFN-α-2b单用并未显示出疗效；13cRA单用及其与IFN-α-2b联合应用均显示出抑制肿瘤生长效应，其作用机制可能为通过下调Cyclin D1的表达而抑制细胞增殖或者激活caspase-9诱导凋亡。

HR-1 Mice: A New Inflammatory Acne Mouse Model.

BackgroundThere is no appropriate in vivo animal model that reflects the inflammatory response of human acne.ObjectiveThis study investigated the effect of Propionibacterium acnes on the development of inflammatory acne-like lesions in four mouse strains with different degrees of immune response for the development of an optimal mouse model of inflammatory acne.MethodsHuman P. acnes suspensions (108 and 109 colony forming unit [CFU]/µl) were injected into the backs of HR-1, BALB/c, vitamin D receptor-knockout (VDR k/o), and severe combined immunodeficiency disease mice. Inflammation levels were evaluated two weeks after injection of P. acnes suspensions. In addition, histopathological examination and immunohistochemical staining of the expressions of inflammatory biomarkers (i.e., CD4+/CD8+ T lymphocytes, neutrophils, myeloperoxidase, interleukin-1β, matrix metalloprotease (MMP)-2, MMP-3, MMP-9, toll-like receptor (TLR)-2, LL-37, and integrin α6) were performed on tissue specimens.ResultsThe HR-1 mouse strain exhibited the most remarkable inflammatory reaction with epithelial proliferation and microcomedone-like cyst formation. HR-1 mice also demonstrated aberrant integrin expression in the epidermis around both inflamed lesions and newly formed microcomedones. These findings were more prominent in the group receiving 109 CFU/µl P. acnes than 108 CFU/µl. MMP-9 expression in HR-1 mice was also upregulated around the microcomedone-like cysts. Finally, expression levels of TLR-2 and LL-37 were higher in HR-1 and BALB/c mice than the VDR k/o and SCID mice strains.ConclusionP. acnes induces acneiform inflammation with small microcomedones in HR-1 mice. Therefore, the HR-1 mouse strain represents a good candidate for the development of a new inflammatory acne mouse model.

Safety of attenuated smallpox vaccine LC16m8 in immunodeficient mice.

Freeze-dried live attenuated smallpox vaccine LC16m8 prepared in cell culture has been the sole smallpox vaccine licensed in Japan since 1975 and was recently recommended as a WHO stockpile vaccine. We evaluated the safety of recently remanufactured lots of LC16m8 using a series of immunodeficient mouse models. These models included suckling mice, severe combined immunodeficiency disease (SCID) mice, and wild-type mice treated with cyclosporine. LC16m8 showed extremely low virulence in each of the three mouse models compared with that of its parental strains, Lister and LC16mO. These results provide further evidence that LC16m8 is one of the safest replication-competent smallpox vaccines in the world and may be considered for use in immunodeficient patients.

N-(4-18F-Fluorobenzoyl)Interleukin-2 for PET of Human-Activated T Lymphocytes

Interleukin-2 (IL2) binds with high affinity to the IL2 receptors overexpressed on activated T lymphocytes in various pathologic conditions. Radiolabeling of IL2 with a positron-emitting isotope could provide a tool for noninvasive PET of activated T cells in immune-mediated diseases. We report the labeling of IL2 with N-succinimidyl 4-(18)F-fluorobenzoate ((18)F-SFB) for the synthesis of N-(4-(18)F-fluorobenzoyl)interleukin-2 ((18)F-FB-IL2) and the in vitro and in vivo evaluation of this novel radiopharmaceutical for the detection of IL2 receptor-positive cells by PET. (18)F-SFB was efficiently prepared using a 3-step radiochemical pathway. Purified (18)F-SFB was incubated with IL2 in borate buffer (pH 8.5) and ethanol at 50°C for 10 min. (18)F-FB-IL2 was purified by reversed-phase high-performance liquid chromatography. As in vitro quality controls, a biologic binding assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis, mass spectrometry, and 3-chloroacetic acid precipitation stability tests were performed. Biodistribution studies were performed in BALB/c mice to evaluate the distribution of the tracer in healthy animals. PET experiments were performed in severe combined immunodeficiency disease mice inoculated with phytohemoagglutinin-activated human peripheral blood mononuclear cells (hPBMc). Whole-body images were acquired 30 min after injection of 5-15 MBq of (18)F-FB-IL2. (18)F-SFB was produced with a 34%-38% radiochemical yield. The radiochemical purity after solid-phase extraction purification ranged from 93% to 96%. Conjugation of (18)F-SFB to IL2 yielded (18)F-FB-IL2 as the major product. The radiochemical yield of (18)F-FB-IL2 after high-performance liquid chromatography purification was 25%-35% based on (18)F-SFB. (18)F-FB-IL2 was stable in plasma at 37°C and capable of stimulating T cells to an extent similar to native IL2. A biodistribution study showed highest tracer uptake in the kidneys and bladder due to rapid renal clearance of the tracer. Small-animal PET images showed binding of (18)F-FB-IL2 to activated hPBMc proportional to the number of injected cells. We report the successful labeling of IL2 with (18)F for PET of activated T lymphocytes. (18)F-FB-IL2 is stable, is biologically active, and allows in vivo detection of activated T lymphocytes.

Inhibition of Primary Effusion Lymphoma Engraftment in Scid Mice by Morpholino Oligomers against Early Lytic Genes of Kaposi'S Sarcoma-Associated Herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several malignant diseases, including Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease. The objectives of this study were to investigate the use of peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) against KSHV early lytic genes and to assess their efficacy in severe combined immunodeficiency disease (SCID) mice against PEL engraftment. PPMOs are short, single-stranded DNA analogues that contain a backbone of morpholine rings and phosphorodiamidate linkages and have high delivery efficiency into cells. PEL cells were treated with PPMOs against viral interferon regulatory factor 1 (vIRF-1) and expression of vIRF-1 was analysed. PPMOs against vIRF-1 and viral interleukin-6 (vIL-6) were evaluated against PEL cell engraftment in SCID mice. The PPMOs were incubated with BCBL-1 cells and then introduced into the peritoneal cavities of SCID mice, followed by 9 more doses of PPMOs administered at 2-day intervals. At weeks 3 and 9 after BCBL-1 delivery, peritoneal lavage was collected and the ratio of PEL cells among total cells was determined by flow cytometry analysis. Treatment of PEL cells with PPMOs against vIRF-1 led to a reduction of vIRF-1 expression in a dose-dependent manner. Reduction of vIRF-1 expression resulted in higher levels of cellular interferon regulatory factor 3 and of signal transducer and activator of transcription 1. SCID mice receiving a PPMO against vIL-6 had no engraftment of PEL cells and remained healthy throughout the 120-day study. This study showed that PPMOs can be effective antiviral agents against KSHV. Blocking the expression of early lytic genes might be beneficial for the control of KSHV-associated malignant diseases.

A Novel Nonoperative Orthotopic Colorectal Cancer Murine Model Using Electrocoagulation

Orthotopic mouse models of human colorectal cancer represent an important in vivo tool for testing chemotherapeutic agents and studying intraluminal factors that may alter the growth of cancers. Currently the orthotopic mouse models of colorectal cancer require either an operative procedure or creation of colitis to implant the cancer cells in rectum. We have developed a nonoperative, minimally invasive technique to create a true orthotopic colon cancer mouse model. We used human (LS 174T and HT-29) and murine (CRL-2638 and CRL-2639) colon cancer cell lines. Low-dose mucosal coagulation was performed transanally using a specially designed electrode at 1 to 3 predetermined points, 2 to 3 cm from the anus, followed by a transanal instillation of tumor cells (1 × 10(6) cells) in 12 female nude or severe combined immunodeficiency disease mice for each of the 4 groups (n = 48, plus 16 controls). Mouse colonoscope (Coloview) and microCT were used to follow tumor growth. Four mice from each group were euthanized at 1, 2, and 3 weeks. Tumors were detected in 12 of 12 of the CRL-2638, 11 of 12 of the CRL-2639, 7 of 12 of the HT-29 and 12 of 12 mice in the LS 174T groups. Histopathologic evaluation showed that the tumors grew from the colonic mucosa. CRL-2638 and LS 174T exhibited better implantation, faster tumor growth, larger tumor volume, and earlier metastases. MicroCT detected tumors larger than 3 mm, and the colonoscopy detected tumors larger than 1 mm. Our mouse model is minimally invasive and easy to create and overcomes the limitations of existing models while mimicking the human disease in terms of morphology and biologic behavior. This model opens the doors for colon cancer oncologic studies in an animal model that were previously not possible.

5-Aminolevulinic acid-induced fluorescence diagnosis of pleural malignant tumor

Background It is known that endogenously synthesized protoporphyrin IX (PpIX) following the administration of 5-aminolevulinic acid (5-ALA) is an effective photosensitizer for photodynamic diagnosis (PDD). We tested in vivo and in vitro susceptibility of human lung cancer and mesothelioma cells to photodynamic diagnosis (PDD) using 5-aminolevulinic acid (5-ALA) as a photosensitizer. Methods Human lung cancer cell lines A549, Ma44-3, FT821 and human mesothelioma cell lines MSTO-211H, NCI-H290, Y-MESO-14 were incubated with 0.03% 5-ALA for 4 h. After incubation, protoporphyrin IX (PpIX) fluorescence was detected using a fluorescence microscope. Pleural carcinosis was induced in severe combined immunodeficiency disease mice using the previous cell lines to test the efficacy of PDD in vivo. The mice were sacrificed 4 h after oral administration of 400 mg/kg of 5-ALA. We counted the visible tumors under white light then fluorescence light. Results In vitro, clear red fluorescence was observed in all cell lines. The mean fluorescence intensity was stronger in A549 and FT821 cells than Ma44-3 cells (165.59 ± 26.49, 157.62 ± 18.93 vs. 104.01 ± 17.58). Also, MSTO-211H and NCI-H290 cells had stronger fluorescence intensity than Y-MESO-14 cells (142.51 ± 26.85, 165.16 ± 12.91 vs. 92.31 ± 8.69). In vivo, the tumor detection rate of fluorescence diagnosis was 1.1–4.5 times higher than that of white light. The mean number of metastases detected by the PDD was significantly higher than that of white light for FT821 ( p = 0.004), Ma44-3 ( p = 0.006) and Y-MESO-14 cell lines ( p = 0.005), but not for A549, NCI-H290 and MSTO-211H cell lines. Small lesions were detected by fluorescence diagnosis even though the lesions were invisible macroscopically under white light. Conclusion Our results suggest the possibility of clinical application of fluorescence diagnosis with intrapleural malignant tumors.

Efficient Inhibition of Human B-cell Lymphoma in SCID Mice by Synergistic Antitumor Effect of Human 4-1BB Ligand/Anti-CD20 Fusion Proteins and Anti-CD3/Anti-CD20 Diabodies

Here we constructed and produced a recombinant human 4-1BB ligand (4-1BBL)/anti-CD20 fusion protein and examined its antitumor activity, alone and in combination with an anti-CD3/anti-CD20 bispecific diabody. The 4-1BBL/anti-CD20 fusion protein retained both the costimulatory activity of 4-1BBL on T cells and the tumor targeting ability of CD20 antibody on B cells. The fusion protein bound as efficiently to 4-1BB- and CD20-positive cells as its respective parental antibodies, and was capable of cross-linking human T lymphocytes and CD20-positive tumor cells. Combination treatment with 4-1BBL/anti-CD20 fusion protein and anti-CD3/anti-CD20 diabody led to significantly increased T-cell cytotoxicity to human B-lymphoma cells in vitro and drastically more potent tumor inhibitory activity in vivo in xenografted B-cell lymphoma in severe combined immunodeficiency disease mice. Mechanistic studies revealed that the combination treatment remarkably inhibited apoptosis of human peripheral blood lymphocytes, accompanied by upregulation of Bcl-XL and Bf1-1, perforin and granzyme B mRNA, and increased interleukin-2 production. Taken together, these results suggest that targeted delivery of 4-1BBL to the tumor site, when combined with anti-CD3/anti-CD20 diabody, could strongly potentiate the antitumor activity of the diabody, thus may have significant clinical application in the treatment of human CD20-positive B-cell malignancies.

Human tissue-specific microenvironment: An essential requirement for mouse models of breast cancer

An ideal mouse model should closely mimic a clinical situation. However, for most models available, this is not the case since clinical trials frequently fail to reproduce the highly encouraging therapeutic results obtained from pre-clinical studies performed using mouse models. In this study, in the process of extending the application of our previously established breast tissue-derived orthotopic and metastatic (BOM) model, the human breast cancer cell line MDA-MB-231 failed to exhibit any osteotropic features that differed from previous studies. Our observations suggest that a human tissue-specific microenvironment could be an essential requirement for a successful mouse model of breast cancer. Here, multiple in vivo breast cancer models were used to confirm this hypothesis. Human breast tissue and cancellated bone were transplanted subcutaneously to female severe combined immunodeficiency disease (SCID) mice by different assemblies, to build several mouse models termed 'breast-breast', 'breast-bone', 'bone-bone', 'MFP (mouse mammary fat pad)-bone', and 'MFP-breast' models. Two human breast cancer cell lines, MDA-MB-231 and MDA-MB-231BO, and the mouse breast cancer cell line TM40D were used. All cancer cells were labeled with GFP for gross observation. In addition, transplanted human tissues and various mouse tissues including bone, lung, liver, mesentery were examined microscopically. Based on morphological, immunohistochemical, and enzymohistochemical evidence obtained from several comparative experiments in 'breast-breast', 'breast-bone' and 'bone-bone' models, the BOM model was proved to be feasible and reliable. The organ tropism of the breast cancer cell line, which was derived from a mouse model by intracardiac inoculation in a pure mouse microenvironment, was reconsidered. The behavior of breast cancer cells in the mouse model was altered in response to the varying microenvironment. The results in this study suggest the human tissue-specific micro-environment is most likely an essential requirement in mouse models of breast cancer.

Cell-penetrating d-Isomer Peptides of p53 C-terminus: Long-term Inhibitory Effect on the Growth of Bladder Cancer

To investigate whether a single application of the membrane-permeable D-isomer of the p53 C-terminus connected with a retro-inverso version of the NH(2)-terminal 20-amino acid peptide of the influenza virus hemagglutinin-2 protein (riHA2) inhibited the growth of bladder cancer cells. The transduction of p53 using poly-arginine is useful for targeting and suppressing the growth of bladder cancer cells. However, the protein's intracellular half-life is short, and repeated application is necessary to achieve an anti-tumor effect. The p53 carboxyl-terminal peptides covalently coupled with cell-penetrating peptides were synthesized with D- or L-amino acids. Moreover, the peptides were connected with riHA2 by a disulfide bridge. Human bladder cancer cell lines were incubated with each peptide and cell viability was assessed with the WST assay. Apoptotic cells were confirmed by Hoechst and active capase-3 staining. The p53 peptides were injected into severe combined immunodeficiency disease mice transplanted with J82 cells to investigate their anti-tumor effect on bladder tumors. A survival curve was plotted using the Kaplan-Meier method. A single application of cell-penetrating D-isomer peptides of the p53 C-terminus connected with riHA2 (d11R-p53C'-riHA2 and dFHV-p53C'-riHA2) inhibited the growth and induced the apoptosis of bladder cancer cells. The tumor-bearing mice treated only with vehicle had a mean survival time of 12 days, whereas treatment with d11R-p53C'-riHA2 resulted in a long-term survival rate of 50%. Peptide transduction therapy using the D-isomer p53 C-terminal peptide with riHA2 may be an innovative method for the treatment of bladder cancer.

Fluorescence diagnosis of lymph node metastasis of lung cancer in a mouse model

Fluorescence diagnosis of lymph node metastasis of non-small cell lung cancer has not been reported previously. This study was conducted to evaluate the feasibility of fluorescence detection using 5-aminolevulinic acid and mono-L-aspartyl chlorine e6 for lymph node metastasis in a lung cancer mouse model. Human lung cancer cell line Ma44-3, which forms metastatic lymph nodes in the mediastinum, was injected into the left lungs of 6 severe combined immunodeficiency disease mice. Two weeks after implantation, 2 groups of 3 mice received oral administration of 5-aminolevulinic acid (100 mg/kg) or intraperitoneal administration of mono-L-aspartyl chlorine e6 (5 mg/kg). Both lungs and mediastinal organs were removed en-bloc and illuminated with blue light (405 nm) to evaluate the detectability of lung tumors and metastatic lymph nodes in the mediastinum. These organs were evaluated histopathologically. Clear red fluorescence was observed in the lung tumors of all mice. Metastatic lymph nodes had formed in 5 of 6 mice and were detected by fluorescent detection in all 5 mice even though one of the lymph nodes was invisible macroscopically under white light. In conclusion, fluorescence diagnosis of lymph node metastasis is possible in a mouse model. The successful result with small lymph node metastasis suggests the possibility of clinical application.

E1A, E1B double-restricted replicative adenovirus at low dose greatly augments tumor-specific suicide gene therapy for gallbladder cancer

Combination therapy with replicative oncolytic viruses is a recent topic in innovative cancer therapy, but few studies have examined the efficacy of oncolytic adenovirus plus replication-deficient adenovirus carrying a suicide gene. We aim to evaluate whether an E1A, E1B double-restricted oncolytic adenovirus, AxdAdB-3, can improve the efficacy for gallbladder cancers (GBCs) of the replication-deficient adenovirus-based herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) therapy directed by the carcinoembryonic antigen (CEA) promoter. Cytopathic effects of AxdAdB-3 plus AxCEAprTK (an adenovirus expressing HSVtk directed by CEA promoter) or AxCAHSVtk (an adenovirus expressing HSVtk directed by a nonspecific CAG promoter) with GCV administration were examined in several GBC lines and normal cells. Efficacy in vivo was tested in severe combined immunodeficiency disease mice with GBC xenografts. Addition of AxdAdB-3 (1 multiplicity of infection, MOI) significantly enhanced the cytopathic effects of AxCEAprTK (10 MOI)/GCV on GBC cells. The augmented effect was attributable to the replication of the AxCEAprTK and also to the enhanced CEA promoter activity, which was presumably transactivated by E1A. In normal cells, AxdAdB-3 (20 MOI) plus AxCEAprTK (200 MOI)/GCV was not cytopathic, whereas AxdAdB-3 (1 MOI) plus AxCAHSVtk (10 MOI)/GCV was significantly toxic. Low-dose AxdAdB-3 (2 x 10(7) PFU, plaque-forming unit) plus AxCEAprTK (2 x 10(8) PFU)/GCV significantly suppressed the growth of GBC xenografts as compared with either AxdAdB-3 (2 x 10(7) PFU)/GCV or AxCEAprTK (2 x 10(9) PFU)/GCV alone. E1A, E1B double-restricted replicating adenovirus at low dose significantly augmented the efficacy of CEA promoter-directed HSVtk/GCV therapy without obvious toxicity to normal cells, suggesting a potential use of this combination for treating GBC and other CEA-producing malignancies.

Mammosphere culture of metastatic breast cancer cells enriches for tumorigenic breast cancer cells

IntroductionThe identification of potential breast cancer stem cells is of importance as the characteristics of stem cells suggest that they are resistant to conventional forms of therapy. Several techniques have been proposed to isolate or enrich for tumorigenic breast cancer stem cells, including (a) culture of cells in non-adherent non-differentiating conditions to form mammospheres and (b) sorting of the cells by their surface phenotype (expression of CD24 and CD44).MethodsWe have cultured metastatic cells found in pleural effusions from breast cancer patients in non-adherent conditions without serum to form mammospheres. Dissociated cells from these mammospheres were used to determine the tumorigenicity of these cultures. Expression of CD24 and CD44 on uncultured cells and mammospheres derived from the pleural effusions was documented.ResultsWe found that the majority (20/27) of the pleural effusions tested contained cells capable of forming mammospheres of varying sizes that could be passaged. After dissociation and plating with serum onto adherent dishes, the cells can differentiate, as determined by the increased expression of cytokeratins and MUC1. Analysis of surface expression of CD24 and CD44 on uncultured cells from 21 of the samples showed that the cells from some samples separated into two populations, but some did not. The proportion of cells that could be considered CD44+/CD24low/- was highly variable and did not appear to correlate with the ability to form the larger mammospheres. Of eight pleural effusion mammospheres tested in severe combined immunodeficiency disease (SCID) mice, four were found to induce tumours when only 5,000 or fewer cells were injected, whereas the same number of uncultured cells did not form tumours. The ability to induce tumours appeared to correlate with the ability to produce the larger mammospheres. Uncultured cells from a highly tumorigenic sample (PE14) were uniformly negative for surface expression of both CD24 and CD44.ConclusionThis paper shows, for the first time, that mammosphere culture of pleural effusions enriches for cells capable of inducing tumours in SCID mice. The data suggest that mammosphere culture of these metastatic cells could provide a highly appropriate model for studying the sensitivity of the tumorigenic 'stem' cells to therapeutic agents and for further characterisation of the tumour-inducing subpopulation of breast cancer cells.