Original Patient Tumor Research Articles

A clinically relevant orthotopic xenograft model of ependymoma that maintains the genomic signature of the primary tumor and preserves cancer stem cells in vivo

Limited availability of in vitro and in vivo model systems has hampered efforts to understand tumor biology and test novel therapies for ependymoma, the third most common malignant brain tumor that occurs in children. To develop clinically relevant animal models of ependymoma, we directly injected a fresh surgical specimen from a 9-year-old patient into the right cerebrum of RAG2/severe complex immune deficiency (SCID) mice. All five mice receiving the initial transplantation of the patient tumor developed intracerebral xenografts, which have since been serially subtransplanted in vivo in mouse brains for 4 generations and can be cryopreserved for long-term maintenance of tumorigenicity. The xenograft tumors shared nearly identical histopathological features with the original tumors, harbored 8 structural chromosomal abnormalities as detected with spectral karyotyping, maintained gene expression profiles resembling that of the original patient tumor with the preservation of multiple key genetic abnormalities commonly found in human ependymomas, and contained a small population (<2.2%) of CD133(+) stem cells that can form neurospheres and display multipotent capabilities in vitro. The permanent cell line (BXD-1425EPN), which was derived from a passage II xenograft tumor and has been passaged in vitro more than 70 times, expressed similar differentiation markers of the xenograft tumors, maintained identical chromosomal abnormalities, and formed tumors in the brains of SCID mice. In conclusion, direct injection of primary ependymoma tumor cells played an important role in the generation of a clinically relevant mouse model IC-1425EPN and a novel cell line, BXD-1425EPN. This cell line and model will facilitate the biological studies and preclinical drug screenings for pediatric ependymomas.

Abstract A25: Establishment and characterization of primary human Biomerk Tumorgraft™ Models: Application to oncology drug development

Abstract One of the key challenges facing oncology drug development is the high attrition rates of compounds that enter the drug development pipeline, where very few achieve successful approval and marketing. Champions Biotechnology, in an effort to enhance the value of preclinical compounds and accelerate oncology drug development, has developed a novel preclinical platform derived from Biomerk Tumorgraft™ models; an innovative approach that utilizes the implantation of primary human tumors in immune-deficient mice in a manner that preserves the biological properties of the original human tumor. Biomerk Tumorgrafts™ differ from traditional xenograft models in that they are not passaged in tissue culture, and are instead exclusively passaged in vivo. Additionally, the Biomerk Tumorgraft™ models: (a) maintain the fundamental genotypic features of the original cancer, (b) represent the genetic heterogeneity of the cancer, and (c) do not change over several passages. Champions has established a bank of over 200 Tumorgraft models encompassing all major solid tumor types, all of which have been extensively characterized. Here we describe a panel of NSCLC lung, colon, and pancreatic tumorgraft models including gene expression profiling, mutational analyses and responses to standard of care agents. Of the NSCLC lung models, 6/8 demonstrated resistance to Erlotinib with 2/8 demonstrating an intermediate response, 2/6 also possessed a mutant K-ras or a mutant EGFR. Similarly, 3/6 K-ras mutant colon Tumorgraft models demonstrated sensitivity to irinotecan. In the case of the pancreatic models, 7/8 showed resistance to Erlotinib with the remaining model showing only a modest response. Through Champions' Personalized Oncology Services, we have also shown a high correlation between the Tumorgraft response and the clinical response. Together, these results demonstrate that each model differs with respect to its genetic composition and response to standard agents, and thus represents the efficacy of a compound on an individual patient. Given the diversity of models, the maintenance of the genomic and phenotypic characteristics of the original patient tumor, and the correlation between Tumorgraft and clinical responses, Champions Biomerk Tumorgraft™ models represent a novel preclinical in vivo platform capable of optimizing drug regimens for cancer patients and predicting the clinical effectiveness of preclinical drug candidates. Overall, the application of Biomerk Tumorgraft™ models has the potential to accelerate and enhance oncology drug development. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A25.

Abstract A173: Profiling of targeted anticancer agents in a panel of 48 patient-derived xenografts using a clonogenic assay

Abstract Novel experimental and approved cancer therapeutics target proteins with key functions in mitosis regulation, cell survival and migration, protein turnover or angiogenesis. These targets include receptor tyrosine (Y) kinases like EGF-R, PDGF-R, VEGF-R, c-KIT and FLT-3, serine-threonine (S/T) specific kinases like B-raf and Aurora A/B (ARK), mitotic kinesins like Eg5 (KSP), the chaperone Hsp90 and traditional targets, namely topoisomerase I / II and ß-tubulin. At Oncotest, a unique profiling screen for anticancer agents using a clonogenic assay using a panel of 48 proprietary solid tumor xenografts is routinely done. The xenograft models in general were established directly from patient tumor material, representing all major tumor histotypes (non-small cell lung cancer / NSCLC, pancreatic, prostate, colon, gastric, breast, ovarian and renal cancer, melanoma and sarcoma) as well as niche tumors (pleuramesothelioma, bladder and head and neck cancer). After an initial profiling screen in the 48 tumor panel, follow-up testing in defined tumor histotypes are of high value for hypothesis generation including selection of a clinical development strategy. Here, activity profiles for inhibitors targeting EGF-R (Erlotinib), Aurora A/B (VX-680), multiple S/T and Y kinases (Sunitinib, Sorafenib), Eg5 (Ispinesib) and tububiln (Vincristine), Hsp90 (17-DMAG) and topoisomerase I (SN-38, active metabolite of Irinotecan) are discussed. Erlotinib displayed a diverse pattern of activity with a mean IC50 = 20.7µM. Selectivity was studied in an extended panel of NSCLC with tumors showing activated EGF-R being mainly most sensitive. The spectrum-kinase inhibitors Sunitinib and Sorafenib displayed weak selectivity with mean IC50 values of 7.9µM and 11.8µM, respectively. The Hsp90 inhibitor 17-DMAG, the topoisomerase I inhibitor SN-38 and Vincristine were highly potent with mean IC50 values of 28nM, 45nM, and 100nM, respectively. For 17-DMAG and SN-38 as examples, tumor xenograft models selected based on clonogenic assay data responded very well in in vivo efficacy tests. Further evaluation of Vincristine in an extended melanoma panel revealed a strong correlation of sensitivity with a B-raf wild type genotype and resistance with the B-raf V600E mutation. This result was described recently for melanoma patients with B-raf mutations, showing a diminished duration of response to treatment with Dacarbazine, Vincristine, Bleomycin, Lomustine, and human leukocyte interferon. In conclusion, the Oncotest clonogenic assay by retaining important characteristics of the original patient tumor is of high value for profiling of traditional cytotoxic as well as new targeted cancer agents. By revealing diverse activity and resistance patterns, molecular characterization data including gene mutations and expression are guiding the selection of tumor entities and patients likely to respond to therapy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A173.

Establishment and characterization of patient tumor-derived head and neck squamous cell carcinoma xenografts

The overall purpose of this study was to establish human head and neck squamous cell carcinoma (HNSCC) xenografts in mice by transplantation of surgical tumor tissue and to characterize the growth, histologic and vascular properties of these xenografts. Primary surgical specimens of HNSCC were xenografted into eight-to-twelve week old severe combined immunodeficiency (SCID) mice. Histologic features of primary HNSCC specimens, initial and established xenografts were compared for tumors established from three different head and neck subsites, namely, oral cavity, larynx and base of tongue(one tumor per site). Growth rates of xenografts were compared along with magnetic resonance imaging (MRI) measures of tumor vascularity and correlative CD31 immunostaining. Initial and established xenografts from all three sites demonstrated a squamous phenotype similar to the original patient tumor histology. Established xenografts of oral cavity and larynx exhibited increased keratinization (H&E) compared to initial xenografts and the primary tumor. No differences in tumor growth rates were observed between established xenografts from the different subsites. Xenografts established from SCC of the larynx exhibited increased microvessel density and lumen area (CD31 staining) along with enhanced permeability to the MR contrast agent compared to oral cavity and base of tongue tumors. Our results show that the combination of non-invasive imaging along with histologic evaluation of patient tumor xenografts offers a valuable platform for preclinical investigations in head and neck cancer. However, it is important to recognize the influence of tumor-host interactions on the histologic phenotype of transplanted tumors.

Characterization of a subpopulation of colon cancer cells with stem cell‐like properties

The biology of the normal colonic mucosa suggests that colon cancer originates from normal colon stem cells. CD44 cancer stem cells have been identified in breast and prostate cancer, and we therefore examined whether CD44 similarly identified colon cancer stem cells. Initial assays found CD44(hi) colon tumor cells to have enhanced soft agar colony-forming ability. Subsequently, CD44(hi) cells isolated from 4 primary colon adenocarcinoma xenografts were found to be highly tumorigenic in immune deficient mice. CD44(hi) cells consistently formed tumors with 1,000 cells, and in multiple experiments, as few as 10 and 100 CD44(hi) cells formed tumors in 7/10 and 21/28 mice, respectively. In contrast, CD44(-) colon tumor cells were either nontumorigenic or 10-50-fold less tumorigenic. CD44(hi) cells could be serially passaged up to 4 times in vivo, suggesting self-renewal capacity, and formed tumors that recapitulated the heterogeneity of the original patient tumor. CD44(hi) cells were significantly enriched for nuclear activated beta-catenin, a key element in normal stem/progenitor cells and in early colon tumor progression. Bromodeoxyuridine (BrdU) labeling studies indicated that CD44(hi) cells divide slowly relative to the CD44(-) cells, suggesting their tumorigenicity is not simply due to faster proliferation. Aldehyde dehydrogenase (ALDH) sort further increased the tumorigenicity of CD44(hi) cells from 2/2 patient tumors, but CD133 tumor cells in our hands did not have increased tumorigenicity. Our observations indicate that CD44 is a marker of stem-like cells in colon cancer, and support the use of additional markers to further purify colon cancer stem cells.

Granulocyte Colony-Stimulating Factor and Granulocyte-Macrophage Colony-Stimulating Factor Promote Malignant Growth of Cells from Head and Neck Squamous Cell Carcinomas In vivo

Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are used to ameliorate cancer therapy-induced neutropenia and mucositis. Yet, first data in head and neck squamous cell carcinoma (HNSCC) indicate an impaired long-term prognosis on G-CSF treatment, and previous studies showed a contribution of both factors to the progression of human epithelial tumors. Therefore, we investigate the role of G-CSF and GM-CSF in progression of tumor cells from human HNSCC. Both factors stimulated proliferation and migration of tumor cell lines established from patient tumors expressing G-CSF and GM-CSF and/or their receptors. Blockade of G-CSF and GM-CSF inhibited tumor cell invasion in a three-dimensional organotypic culture model. The contribution of both factors to tumor malignancy was further confirmed in nude mouse transplants in vivo. Invasive and malignant growth yielding a similar tumor phenotype as the original patient tumor was exclusively observed in G-CSF- and GM-CSF-expressing tumors and was associated with enhanced and persistent angiogenesis and enhanced inflammatory cell recruitment. Although factor-negative tumors grew somewhat faster, they were characterized by lack of invasion, reduced and transient angiogenesis, and large necrotic areas. These data provide evidence for a progression-promoting effect of G-CSF and GM-CSF in human HNSCC and suggest further detailed evaluation of their use in the therapy of these tumors.

Karyotypic analysis of a heterogeneous human transitional cell carcinoma of the bladder

The UCRU-BL-17 (BL-17) series of xenografts, tissue culture sublines, and cloned cell lines (Fig. 1) shows a range of heterogeneous growth characteristics both in vitro and in vivo (Table 1) and represents a model of human bladder cancer heterogeneity. Cytogenetic analysis was undertaken to determine if specific chromosome changes correlated with particular aspects of the heterogeneous phenotypes. The BL-17 sublines and cloned cell lines shared many common chromosome abnormalites. Indeed, the cloned cell lines showed nearly identical karyotypes despite their marked differences in growth characteristics. Karyotypic evolution with passage through the nude mice was apparent, however. This evolution occurred at the specific chromosome regions of 1p12, 3cen-3p21, and 6cen-6q21. Whether the heterogeneity of karyotype between the BL-17 cell lines resulted from the existence of multiple clones in the original patient tumor or from karyotypic instability through passage in nude mice is uncertain, but in either case the specificity of karyotypic evolution observed suggests that 1p12, 3cen-3p21, and 6cen-6q21 are hotspots for rearrangement in the BL-17 tumor. No specific correlations between chromosome abnormalities and biologic characteristics could be made, but several unique karyotypic features arose in the progression to two of the sublines, BL-17/23α and BL-17/0/X2A, coinciding with a loss of anchorage-independent growth by BL-17/23α and a change in growth in vivo from a solid tumor to a fluid-filled tumor by BL-17/0/X2A.

Characterization of a human endometrial carcinoma cell line producing intraperitoneal tumor growth in immunodeficient mice

Establishment of laboratory models of gynecologic neoplasms provides an important means of studying the biologic characteristics of these tumors. We report a previously uncharacterized human endometrial adenocarcinoma cell line that produces both intraperitoneal and subcutaneous growth in nude mice. The line was derived from a poorly differentiated endometrial cancer and has been carried in continuous tissue culture for greater than 100 passages. Doubting time in culture is approximately 48 hr. Antigenic phenotyping against a panel of murine monoclonal antibodies by resetting cell surface assay on live cells or peroxidase assay on fixed cells has shown reactivity with a number of determinants, including MH99, MT334, MQ49, and the blood group antigens F3, 118, and 41–83. Cytogenetically, the line displays an aneuploid human karyotype with several chromosomal rearrangements and deletions. When injected intraperitoneally into nude mice, animals develop intraperitoneal nodules and ascites and succumb with wasting in 30–40 days. The intraperitoneal tumor has been passaged multiple times in nude mice by direct transfer of ascites. Subcutaneous injection of tumor cells produces nodules that grow at a reproducible rate. By light and electron microscopy, the nude mouse tumor is a poorly differentiated adenocarcinoma, similar to the original patient's tumor. It expresses both estrogen and progesterone receptors. CA 125 is not elevated in the serum of animals with tumor implants. The line appears to be cisplatin sensitive as determined by rates of growth of subcutaneous nodules. This cell line may be useful in studying the in vitro and in vivo properties of human endometrial carcinoma.

Effects of steroid hormone therapy on primarily xenotransplanted human colorectal adenocarcinomas.

Primary xenotransplantation of six different human colorectal adenocarcinomas onto nude mice yielded a mean tumor take of 85%. Administration of steroid hormones induced tumor remissions in some cases. Neither the stage of the original patient's tumors nor their hormone receptor content seemed to be related to the result of the hormone therapies. It is concluded that some colorectal cancers can be treated as hormone-sensitive tumors.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.