Orthotopic and ectopic organ environments differentially influence the sensitivity of murine colon carcinoma cells to doxorubicin and 5-fluorouracil.

We determined the effects of organ environment on the response of murine CT-26 colon carcinoma cells to 2 structurally and pharmacologically distinct chemotherapeutic agents. CT-26 cells were injected i.v. (to produce lung lesions), s.c., into the cecal wall, and into the spleen (to produce spleen and liver lesions). Doxorubicin (DXR) at 10 mg/kg, 5-fluorouracil (5-FU) at 20 mg/kg, or saline (control) was injected intravenously on different schedules after tumor-cell implantation. The in vivo responses of the tumors growing in the cecum, spleen, liver, lung and subcutis were compared. Colon carcinomas growing in the subcutis were most sensitive to DXR. Tumors growing in the spleen and cecum were most sensitive to 5-FU and less so to DXR. Tumors in the liver were highly resistant to both drugs, whereas experimental lung metastases were sensitive to 5-FU but resistant to DXR. The differential responses of the tumors to the drugs were not due to drug distribution. The level of protein-kinase-C activity was elevated in the spleen, liver and cecum tumors as compared with s.c. tumors and correlated with the in vivo DXR resistance of the tumor cells. This correlation suggested that organ environment may modulate the chemosensitivity of tumor cells, at least in part, by perturbing signal transduction pathways. Collectively, the data indicate that the organ environment has profound effects on the response of tumor cells to chemotherapy. A molecular understanding of this phenomenon should facilitate the design of more effective systemic chemotherapy for cancer metastases.     

Angiogenesis and growth of murine colon carcinoma are dependent on infiltrating leukocytes

We determined whether the angiogenesis and growth of murine colon carcinomas growing in the wall of the cecum is dependent on infiltrating leukocytes. Syngeneic BALB/c or SCID mice were treated with a myelosuppressive, maximally tolerated dose of doxorubicin. Parental or multidrug resistant CT-26 colon carcinoma cells were implanted into the cecal wall 3 days after the second intravenous injection of doxorubicin. Control mice developed large, well-vascularized tumors, whereas doxorubicin-pretreated mice did not. Intravenous injection of spleen cells from normal BALB/c or SCID mice one day prior to tumor cell implantation reversed the decreased vascularity and tumorigenicity. The production of proangiogenic molecules and microvessel density in tumors directly correlated with the number of infiltrating leukocytes, suggesting that tumor-infiltrating leukocytes are essential to angiogenesis of murine colon carcinomas.     

Modulation of tumor cell response to chemotherapy by the organ environment

The outcome of cancer metastasis depends on the interaction of metastatic cells with various host factors. The implantation of human cancer cells into anatomically correct (orthotopic) sites in nude mice can be used to ascertain their metastatic potential. While it is clear that vascularity and local immunity can retard or facilitate tumor growth, we have found that the organ environment also influences tumor cell functions such as production of degradative enzymes. The organ microenvironment can also influence the response of metastases to chemotherapy. It is not uncommon to observe the regression of cancer metastases in one organ and their continued growth in other sites after systemic chemotherapy. We demonstrated this effect in a series of experiments using a murine fibrosarcoma, a murine colon carcinoma, and a human colon carcinoma. The tumor cells were implanted subcutaneously or into different visceral organs. Subcutaneous tumors were sensitive to doxorubicin (DXR), whereas lung or liver metastases were not. In contrast, sensitivity to 5-FU did not differ between these sites of growth. The differences in response to DXR between s.c. tumors (sensitive) and lung or liver tumors (resistant) were not due to variations in DXR potency or DXR distribution. The expression of the multidrug resistance-associated P-glycoprotein as determined by flow cytometric analysis of tumor cells harvested from lesions in different organs correlated inversely with their sensitivity to DXR: increased P-glycoprotein was associated with overexpression ofmdr1 mRNA. However, the organ-specific mechanism for upregulatingmdr1 and P-glycoprotein has yet to be elucidated.     

Inhibition of Epidermal Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor Phosphorylation on Tumor-Associated Endothelial Cells Leads to Treatment of Orthotopic Human Colon Cancer in Nude Mice

The purpose of our study was to determine whether the dual inhibition of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) signaling pathways in tumor-associated endothelial cells can inhibit the progressive growth of human colon carcinoma in the cecum of nude mice. SW620CE2 human colon cancer cells growing in culture and orthotopically in the cecum of nude mice expressed a high level of transforming growth factor alpha (TGF-α) and vascular endothelial growth factor (VEGF) but were negative for EGFR, human epidermal growth factor receptor 2 (HER2), and VEGFR. Double immunofluorescence staining revealed that tumor-associated endothelial cells expressed EGFR, VEGFR2, phosphorylated EGFR (pEGFR), and phosphorylated VEGFR (pVEGFR). Treatment of mice with either 7H-pyrrolo [2,3-d]-pyrimidine lead scaffold (AEE788; an inhibitor of EGFR and VEGFR tyrosine kinase) or CPT-11 as single agents significantly inhibited the growth of cecal tumors (P < .01); this decrease was even more pronounced with AEE788 combined with CPT-11 (P < .001). AEE788 alone or combined with CPT-11 also inhibited the expression of pEGFR and pVEGFR on tumor-associated endothelial cells, significantly decreased vascularization and tumor cell proliferation, and increased the level of apoptosis in both tumor-associated endothelial cells and tumor cells. These data demonstrate that targeting EGFR and VEGFR signaling on tumor-associated endothelial cells provides a viable approach for the treatment of colon cancer.     

In vivo selection of highly metastatic cells from surgical specimens of different primary human colon carcinomas implanted into nude mice

The purpose of these studies was to select and isolate cells with increased liver-metastasizing potential from heterogeneous primary human colon carcinomas (HCCs). Cells derived from a primary HCC classified as Dukes' stage B2 were directly established in culture or were injected into the subcutis, cecum, or spleen of nude mice. Progressively growing tumors were excised, dissociated, and established in culture. Subsequent to implantation into the cecum or spleen of nude mice, cells from all four lines produced only a few liver tumor foci. HCC cells from the few liver metastases were expanded in culture and then injected into the spleen of nude mice to provide a source for further cycles of selection. With each successive in vivo selection cycle, the metastatic ability of the isolated propagated cells increased. Four cycles of selection yielded cell lines with a very high metastatic efficiency in nude mice. In parallel studies using another primary HCC classified as Dukes' stage D, we isolated cell lines that were highly metastatic in nude mice. Successive selection cycles for growth in the liver increased the metastatic properties of the HCC cells, albeit to a lesser extent than it did those of the Dukes' B2 stage HCC. The ability of the HCC cells to produce liver metastases was not due to simple trapping in the liver. In vivo distribution studies using [125I] iododeoxyuridine-labeled tumor cells revealed that, shortly after injection into the spleen, a comparable number of cells with either low or high metastatic properties arrested in the liver. The differences between the low- and high-degree metastatic cells became apparent by 24 h after injection and, by 72 h, only highly metastatic cells survived in the liver. These results demonstrate that hepatic metastasis by HCC cells is a selective process and that the nude mouse model can be useful for isolating highly metastatic HCC cells and for studying the relevant host organ factors that regulate the pathogenesis of metastasis.     

Tumor invasion and extracellular matrix degradative enzymes: regulation of activity by organ factors.

Degradation of the extracellular matrix (ECM) during cellular invasion is important to the formation of tumor metastasis. Several types of ECM degradative enzymes have been implicated in metastatic invasion by malignant cells. These enzymes are regulated by various factors which vary qualitatively and quantitatively between organs. Tissue and plasma activators of zymogens, enzyme inhibitors, and cell surface enzyme receptors directly control localized enzyme activities, and the expression of enzymes is modulated by various cytokines, growth factors and ECM components. Thus, the interaction of tumor cells with an organ environment affects the metastatic behavior of the cells. This organ factor regulation is illustrated using human colon carcinoma cells implanted in different organ sites of nude mice. The cells growing in the cecal wall produced high levels of ECM degradative enzymes and metastasized to lymph nodes and liver. In contrast, enzyme production was repressed in colon carcinoma tumors growing in the subcutis and no metastases were formed.     

Early tumor growth in metastatic organs influenced by the microenvironment is an important factor which provides organ specificity of colon cancer metastasis

We have previously demonstrated that liver metastases in nude mice and lung metastases in nude rats occurred specifically, when KM12SM human colon carcinoma cells were inoculated orthotopically into the cecal wall of nude mice and rats. To clarify the relationship between the tumor growth potential in the metastatic organs and the metastatic organ preference in these two metastatic models, we have evaluated the in vitro cell growth activities affected by the organ conditioned medium (CM) from the liver and lung, and the in vivo growth activities of the ectopic implanted tumors in the liver and lung. The tumorigenicity of the ectopic implanted tumors was 100% in mouse liver, 33% in rat liver, 50% in mouse lung, and 75% in rat lung. The crude liver CM of the animals showed inhibitory activities for KM12SM cell growth in a dosage-dependent manner, and the crude lung CM stimulated KM12SM cell growth. The liver CM of nude mice inhibited the KM12SM cell growth more strongly compared with the CM of nude rats, and the lung CM of nude rats was more strongly stimulated compared with the CM of nude mice. The liver CM of nude mice had non-heparin binding factors, which stimulated or inhibited KM12SM cell growth, in a molecular weight range of 50 to 100 kDa. By contrast, the liver CM of nude rats showed no growth stimulating activity for KM12SM cells. These results suggest that the metastatic organ specificity of KM12SM cells may depend on the early tumor growth influenced by the microenvironment in metastatic organs.     

Intraperitoneal and subcutaneous xenografts of human ovarian carcinoma in nude mice and their potential in experimental therapy

Human ovarian carcinomas (HOC) were established s.c. and i.p. in nude mice and the biological characteristics were investigated for 4 xenografts. HOC8 and HOC18, derived respectively from a primary tumor of the ovary and a pleural effusion (from 2 different patients) were established s.c. in nude mice. HOC10 and HOC22, derived from the ascites of 2 patients, were directly established as ascites after i.p. injection in nude mice. The s.c. and i.p. growth behavior of the 4 HOC lines was investigated. HOC18, HOC8 and HOC22 cells produced progressively growing tumor after s.c. injection but HOC10 ascites would not grow s.c. The cell suspension derived from HOC18 only produced carcinomatosis upon i.p. injection, while HOC8 cells produced both ascites and carcinomatosis. The 2 ascites HOC10 and HOC22 produced ascites in nude mice, but only HOC22 formed i.p. carcinomatosis. Histopathological characteristics of the patients' primary tumors persisted in nude mice, regardless of the site of tumor implantation. DNA histograms of the xenografts closely matched the patients' tumors and remained stable at different passages. Cisplatin, adriamycin and cyclophosphamide given i.v. were tested against HOC8 and HOC18 growing s.c. and HOC22 and HOC10 growing i.p. HOC8 showed a significant response to DDP and almost no sensitivity to ADR and CTX. HOC18 showed only moderate growth delay with all 3 drugs. Mice bearing HOC10 and HOC22 ascites had a prolonged survival time after DDP and ADR treatment.     

Influence of implantation site on growth, antigen expression and metastatic potential of human colonic cancer HT29 and 5583 xenografts in nude mice

In order to study the interaction between tumors and host environmental factors, we xenografted cells from the human colonic carcinoma cell lines HT29 and 5583-S in the subcutis, cecum, spleen and liver of nude mice and compared growth characteristics, metastatic potential and some phenotypic features of the xenografts in these sites. No remarkable differences were observed between the tumors at different inoculation sites in regard of their expression of carcinoembryonic antigen and secretory component or type of mucin produced. Also the proportion of DNA synthesizing cells as determined by bromodeoxyuridine incorporation appeared to be comparable in the studied implantation sites. Local invasive growth characteristics and metastatic potential, however, showed marked differences. Subcutaneous and cecal xenografts frequently showed tumor cell invasion into the surrounding tissue, vasoinvasive growth and discontinuous basement membrane deposition, whereas splenic and hepatic implants demonstrated more encapsulation, no invasion of blood vessels and more continuous basement membrane deposition. Subcutaneous xenografts produced no metastasis. With HT29 cells liver and lymph node metastases occurred frequently from the splenic as well as the cecal xenografts. 5583 cells regularly produced liver metastases from the splenic xenografts, whereas no metastasis from cecal xenografts were observed. We conclude that although the patterns of invasive growth and the metastatic potential differ for various implantation sites, antigen expression and cell kinetic features of tumor implants are hardly influenced by the site of inoculation.     

Selection of metastatic variants from heterogeneous tumor cell lines using the chicken chorioallantoic membrane and nude mouse

The chicken chorioallantoic membrane was used to select variant tumor cell subpopulations from the murine melanoma B16-BL6 and the rat glioma C6 cell lines. Tumor cells were deposited on the chicken chorioallantoic membrane of eggs 10 days postfertilization. Upon hatching, chickens were autopsied, and organs were removed, minced, and implanted s.c. in C57BL/6J mice (for melanoma) or nude mice (for glioma). A glioma growing s.c. from a chicken lung implant metastasized to the liver of the recipient nude mouse, and a melanoma growing s.c. from a chicken liver implant metastasized to the lung of its murine host. The s.c. melanoma contained distinct black and gray areas. Cell lines were established from the s.c. glioma (C6-V-1), from a metastasis of the C6-V-1 tumor (C6-V-2), and from the black and gray regions of the melanoma. Marked differences in lung colonization were seen 14 days after 1 X 10(5) parent BL6, Black, or Gray cultured cells were injected by tail vein into C57BL mice. In four separate experiments, fewer than 15 lung foci per mouse were found when BL6 cells were injected, whereas 100 to several hundred lung melanoma colonies per mouse were observed when Black or Gray cells were inoculated. Four of 18 nude mice bearing the s.c. C6-V-1 glioma developed liver metastases; no metastases have been observed in 15 nude mice bearing the s.c. parent C6 glioma. Significant differences in sensitivities to antineoplastic drugs were demonstrated between parent and variant glioma cell lines. The 33-fold increase in sensitivity to vincristine determined for C6-V-1 cells compared to parent C6 cells was particularly striking. Results suggest that the use of the chicken chorioallantoic membrane in situ, together with the nude mouse, might provide a method suitable for the selection and isolation of aggressive variants in heterogeneous human tumors.     

Multikinase inhibitor regorafenib inhibits the growth and metastasis of colon cancer with abundant stroma

Interaction between tumor cells and stromal cells plays an important role in the growth and metastasis of colon cancer. We previously found that carcinoma‐associated fibroblasts (CAFs) expressed platelet‐derived growth factor receptor‐β (PDGFR‐β) and that PDGFR targeted therapy using imatinib or nilotinib inhibited stromal reaction. Bone marrow‐derived mesenchymal stem cells (MSCs) migrate to tumor stroma and differentiate into CAFs. A novel oral multikinase inhibitor regorafenib inhibits receptor tyrosine kinases expressed on stromal cells (vascular endothelial growth factor receptor 1–3, TIE2, PDGFR‐β, and fibroblast growth factors) and tumor cells (c‐KIT, RET, and BRAF). These molecules are involved in tumor growth, angiogenesis, lymphangiogenesis, and stromal activation. Therefore, we examined whether regorafenib impaired the tumor‐promoting effect of CAFs/MSCs. KM12SM human colon cancer cells alone or KM12SM cells with MSCs were transplanted into the cecal wall of nude mice. Co‐implantation of KM12SM cells with MSCs into the cecal wall of nude mice produced tumors with abundant stromal component and promoted tumor growth and lymph node metastasis. Single treatment with regorafenib inhibited tumor growth and metastasis by inhibiting both tumor cells and stromal reaction. This tumor‐inhibitory effect of regorafenib was more obvious in tumors developed by co‐implanting KM12SM cells with MSCs. Our data suggested that targeting of the tumor microenvironment with regorafenib affected tumor cell–MSC interaction, which in turn inhibited the growth and metastasis of colon cancer.     

Growth and metastasis of tumor cells isolated from a human renal cell carcinoma implanted into different organs of nude mice

The purpose of this study was to determine whether the methods for isolating tumor cells from a human renal cell carcinoma (HRCC) influence the biological behavior of the cancer cells. Renal cell carcinoma obtained from a surgical specimen was dissociated by enzymatic treatment and cells were plated into culture dishes or injected s.c. into the kidney of BALB/c nude mice. The resultant kidney tumor produced liver metastasis and ascites. All tumors growing in nude mice (s.c., kidney, liver, ascites) were also established in culture. The human origin of all five lines was ascertained by karyotypic and isoenzyme analyses. Cells from all lines were injected, s.c., i.p., i.v., intrasplenically, and beneath the renal capsule of nude mice. All the lines were tumorigenic after s.c. or renal subcapsule injection, although the rate of tumor growth varied among the five lines. The metastatic behavior of the HRCC cells was influenced by both the nature of the tumor cells and the route of injection into nude mice. In general, cells derived from the liver metastasis produced more metastases in nude mice than other lines. The lines established in culture from the primary HRCC and the ascites were poorly metastatic. Even with highly metastatic cells, i.v. injection did not yield significant metastasis, but the injection of cells into the renal subcapsule resulted in extensive metastasis to the lungs and in all peritoneal organs. These results indicate that nude mice can be used for the isolation of populations of HRCC cells with different growth and metastatic potential and that, of the organ sites tested, the renal subcapsule is the most advantageous site for implantation of HRCC cells.     

抗大肠癌大鼠单克隆抗体R19的建立和特性

用大鼠杂交瘤技术，将人盲肠癌Ｈｃｅ－８６９３细胞免疫ＬＯＵ／ｃ大鼠，取其脾细胞与大鼠骨髓瘤ＩＲ９８３Ｆ细胞融合，共产生６２０株杂交瘤从中筛选出分泌抗人大肠癌单抗杂交瘤１９５株，阳性率为３１％。其中Ｒ１９株分泌ＩｇＧ２ｂ型抗体，与人大肠癌Ｈｃｅ－８６９３ｔ ＨＴ０２９ｘｌｅｑｋｇｆ 阳性反应，与人鼻咽癌ＣＮＥ２细胞有弱阳性交叉反应，与正常人胚肺２ＢＳ细胞，人肝癌、等共１５种细胞地交叉反应，显著较强的     

A new animal model for human colon cancer metastasis

An animal model for human colon cancer metastasis is described in which spontaneously metastasizing colonic tumors are formed after injection of human colon cancer cells into the cecal wall of young athymic nude mice. Lymphatic and vascular invasion were demonstrated histologically after injection of both well- and poorly-differentiated cell lines, and metastases were found in a pattern similar to that of naturally occurring human colonic cancer. In contrast, little or no visceral organ involvement could be demonstrated after s.c. injection. Cells with increased liver-metastasizing potential were obtained by serial selection in this system. These cells had an enhanced ability to penetrate a reconstituted basement membrane in the presence of partially purified liver extract when compared to lung or brain extracts in a modified Boyden chamber assay. These results demonstrate the ability of human epithelial tumor cells to metastasize reproducibly in an animal model system, which may be useful for studying many aspects of the pathogenesis of cancer metastasis. In addition, it is suggested that local invasion by colon cancer cells may be influenced in part by tissue-specific factors.     

Comparison of the growth and metastasis of four human intestinal tumor cell line xenografts

The growth and metastasis of four human intestinal tumor cell lines: one duodenal adenocarcinoma (HTB-40) and three adenocarcinomas of the colon (CCL-218, CCL-222 and HT-29) have been compared in vitro and in vivo in nude mice. HTB-40 was the fastest growing cell line in vitro with a doubling time (DT) of 14.8 h. CCL-218 and CCL-222 grew more slowly in vitro with doubling times of 21.6 and 22.8 hours, respectively. All three of these tumors grew more slowly in vivo with doubling times ranging from 39.1 h (CCL-218 in male nude mice) to 65.3 h (CCL-222). The growth of CCL-218 cells was significantly slower in female nude mice DT 51.0 h). HT-29 was the slowest growing in vitro (DT 23.8 h) and in vivo (DT about 100 h). HT-29 also showed the greatest discrepancy between its DT measured in vivo as compared to in vitro, suggesting a greater clonogenic cell loss from HT-29 tumors in vivo. Histologic evaluation of these tumors grown subcutaneously in nude mice showed all to be anaplastic and to produce liver micrometastases. However, more extensive abdominal and liver metastases were observed in the nude mice injected with HT-29 cells, and some of these metastases had morphologic features of moderately well-differentiated epithelium. These results indicate the usefulness of the HT-29 tumor cell line as an experimental model of metastasis from a human colonic adenocarcinoma.     

Comparison of the inhibitory effect of the angiogenesis inhibitor,TNP-470, and mitomycin c on the growth and liver metastasis of human colon cancer

Angiogenesis inhibitors have attracted considerable interest. The anti-tumor and anti-metastatic effects of TNP-470, an angiogenesis inhibitor, and mitomycin C (MMC), a representative anti-neoplastic agent, were investigated using a xenotrans-planted human colon cancer, TK-4. Suturing of small pieces of TK-4 tumors to the cecal wall in nude mice (orthotopic transplantation) induced liver metastasis. Mice were randomly divided into 3 groups; a control group given saline solution, a group receiving TNP-470 and a group receiving MMC. TNP-470 was given s.c. on alternate days for 5 weeks from day 10 after cecal transplantation and MMC was administered intraperitoneally (i.p.) once a week from day 10 after cecal transplantation. MMC significantly inhibited cecal tumor growth. In the control group, liver metastases developed in 9 out of 10 mice, including 3 with more than 20 metastatic foci. Liver metastasis also developed in 8 out of 10 mice receiving MMC, 2 of which had many metastases. In contrast, liver metastasis developed in only 2 out of 8 mice in the TNP-470 group and neither of these animals had numerous metastases. © 1995 Wiley-Liss, Inc.     

Vascular endothelial growth factor and enhanced angiogenesis do not promote metastatic conversion of a newly established azoxymethane-induced colon cancer cell line

The organo-specific carcinogen, azoxymethane (AOM), produces colon tumors in mice that share many pathological features with sporadic human colorectal cancer (CRC). An important distinction between AOM-induced CRC and human CRC is lack of mucosal invasion in the murine model. To assess the role of the microenvironment in preventing the invasive phenotype, multiple benign in situ adenocarcinomas were harvested from AOM-treated mice and cultured in vitro. However, tumor cell growth was extremely limiting under standard culturing conditions. Thus, we injected tumor cells directly into nude mice and performed two serial transplants, and successfully explanted a rapidly growing epithelial tumor cell line (AJ02nm(0)). When injected subcutaneously (sc) into nude mice, AJ02nm(0) cells formed well-differentiated adenocarcinomas with minimal tumor invasive capacity. To define whether metastatic and invasive potential were related to lack of angiogenic stimuli, the AJ02nm(0) cells were transfected to overexpress murine vascular endothelial growth factor-164 (VEGF(164)). AJ02nm-VEGF cells produced rapidly growing tumors in nude mice that exhibited extensive pseudo-epithelial ductal architecture and supporting vasculature, but without increased invasive potential compared to controls. The established murine colon epithelial cell line provides a useful experimental model to further elaborate genetic and epigenetic factors that may promote or inhibit colon tumorigenesis and metastasis.     

Pharmacokinetic profile of the microtubule stabilizer patupilone in tumor-bearing rodents and comparison of anti-cancer activity with other MTS in vitro and in vivo

Introduction  Patupilone is a microtubule stabilizer (MTS) currently in clinical development. Here, we evaluate the anti-cancer activity in vitro and in vivo in comparison to paclitaxel and describe the pharmacokinetics (PK) of patupilone in tumor-bearing nude mice and rats. Methods  The potency in vitro of patupilone and two other MTS, paclitaxel and ixabepilone, was determined using human colon carcinoma cell lines with low (HCT-116, HT-29, RKO) and high (HCT-15) P-glycoprotein expression (P-gp), as well as two multi-drug resistance (MDR) model cell pairs, MCF7/ADR and KB-8511 cells and their respective drug-sensitive parental counterparts. The PK of patupilone was investigated in nude mice bearing HCT-15 or HT-29 xenografts and in rats bearing s.c. pancreatic CA20498 tumors or A15 glioma tumors. Anti-cancer activity in vivo was compared to that of paclitaxel using three different human tumor colon models. The retention and efficacy of patupilone was compared in small and large HT-29 xenografts whose vascularity was determined by non-invasive magnetic resonance imaging. Results  Patupilone was highly potent in vitro against four different colon carcinoma cell lines including those showing multi-drug-resistance. In contrast, paclitaxel and ixabepilone displayed significantly reduced activity with markedly increased resistance factors. In both rats and mice, a single i.v. bolus injection of patupilone (1.5–4 mg/kg) rapidly distributed from plasma to all tissues and was slowly eliminated from muscle, liver and small intestine, but showed longer retention in tumor and brain with no apparent elimination over 24 h. Patupilone showed significant activity against three human colon tumor models in vivo, unlike paclitaxel, which only had activity against low P-gp expressing tumors. In HT-29 tumors, patupilone activity and retention were independent of tumor size, blood volume and flow. Conclusions  The high potency of patupilone, which is not affected by P-gp expression either in vitro or in vivo, and favorable PK, independent of tumor vascularity, suggest that it should show significant activity in colorectal cancer and in other indications where high P-gp expression may compromise taxane activity.     

Characterization of an animal model of metastatic colon carcinoma

Although numerous animal tumor models have been used to study colon carcinoma, few display metastatic properties. We have characterized an animal tumor model that has 3 properties essential for the study of metastasis of colon carcinoma cells: epithelial cell origin; a reproducible pattern of metastatic behavior and the ability to be propagated both in vitro and in vivo to facilitate identification of biochemical correlates of metastasis. The K12/TR cell line was derived from a transplantable colon carcinoma induced by dimethylhydrazine in the BD-1X rat strain. Transmission electron microscopy of K12/TR cells demonstrated junctional complexes, desmosomes and surface microvilli characteristic of gastrointestinal epithelial cells. The epithelial cell origin of K12/TR was confirmed by demonstrating the presence of keratin, a marker of epithelial cells, but not vimentin, a constituent of mesenchymal cells. Secretion of CEA and Ca19-9 antigens by K12/TR cells in vitro was below the sensitivity of the assays (1 ng/ml and 6 U/ml respectively). K12/TR cells produced tumors following s.c. injection into syngeneic BD-1X rats, allogeneic RNU/rnuDF rats and xenogeneic CRL:nu/nuBR mice. Macroscopic lung metastases were observed in animals from all 3 groups. Distal lymph node metastases were more frequent in BD-1X rats than in nude rats or mice. The histological appearances of all tumors and metastases were similar, showing a moderate to poorly differentiated glandular carcinoma. Intrasplenic injections of K12/TR cells in nude mice resulted in liver colonization. Preferential growth of tumor cells at sites of trauma was also observed. The results show that the K12/TR system can be used as a model to study metastasis of colon carcinoma cells and may find utility in the testing of chemotherapeutic agents against metastatic lesions.