Improvement of cancer-targeting therapy, using nanocarriers for intractable solid tumors by inhibition of TGF-β signaling

Abstract

Transforming growth factor (TGF)-β plays a pivotal role in regulation of progression of cancer through effects on tumor microenvironment as well as on cancer cells. TGF-β inhibitors have recently been shown to prevent the growth and metastasis of certain cancers. However, there may be adverse effects caused by TGF-β signaling inhibition, including the induction of cancers by the repression of TGF-β-mediated growth inhibition. Here, we present an application of a short-acting, small-molecule TGF-β type I receptor (TβR-I) inhibitor at a low dose in treating several experimental intractable solid tumors, including pancreatic adenocarcinoma and diffuse-type gastric cancer, characterized by hypovascularity and thick fibrosis in tumor microenvironments. Low-dose TβR-I inhibitor altered neither TGF-β signaling in cancer cells nor the amount of fibrotic components. However, it decreased pericyte coverage of the endothelium without reducing endothelial area specifically in tumor neovasculature and promoted accumulation of macromolecules, including anticancer nanocarriers, in the tumors. Compared with the absence of TβR-I inhibitor, anticancer nanocarriers exhibited potent growth-inhibitory effects on these cancers in the presence of TβR-I inhibitor. The use of TβR-I inhibitor combined with nanocarriers may thus be of significant clinical and practical importance in treating intractable solid cancers.

Chemotherapy that uses nanocarriers has been developed to improve the clinical treatment of solid tumors by obtaining high accumulation of drugs in tumor tissues but limited accumulation in normal organs. Doxil (1), a liposomal adriamycin (ADR), is one such drug that has already been used clinically (2). Doxil has exhibited therapeutic effects on some cancers with hypervascular characteristics (3, 4), including Kaposi sarcoma and ovarian cancers. Another promising formulation of nanocarriers is polymeric micelles (5, 6), which are already being used in clinical trials (7, 8).

However, despite the urgent need for effective chemotherapy for intractable solid tumors, including pancreatic adenocarcinoma (9) and diffuse-type gastric carcinoma (10), nanocarriers of any design have not been successful yet in exhibiting significant therapeutic effects on these cancers. Pancreatic cancer is the fourth leading cause of cancer-related death in the United States and the fifth in Japan (9), and the median survival period of patients who suffer from advanced pancreatic adenocarcinoma is still extremely short (≈6 months), despite recent progress in development of conventional chemotherapies (11). Although cancer cells derived from these tumors are sufficiently sensitive in vitro to conventional anticancer agents such as ADR (12), most of these agents have failed to exhibit sufficient therapeutic effects in vivo, regardless of formulation, whether encapsulated in nanocarriers or not. The theoretical basis of the specific accumulation of nanocarriers in tumor tissues is leakiness of tumor vessels to the macromolecular agents, termed the “enhanced permeability and retention (EPR) effect,” which was demonstrated and named by Maeda et al. (13, 14). The major obstacles to treatment of these cancer cells could thus be insufficient EPR effect because of certain characteristics of their cancer microenvironment, including hypovascularity and thick fibrosis (15, 16). However, methods of regulating this effect have not been well investigated.

Transforming growth factor (TGF)-β signaling plays a pivotal role in both the regulation of the growth and differentiation of tumor cells and the functional regulation of tumor interstitium (17). Because TGF-β is a multifunctional cytokine that inhibits the growth of epithelial cells and endothelial cells and induces deposition of extracellular matrix, inhibition of TGF-β signaling in cancer cells and fibrotic components has been expected to facilitate the effects of anticancer therapy. TGF-β binds to type II (TβR-II) and type I receptors (TβR-I), the latter phosphorylates Smad2 and −3. Smad2 and −3 then form complexes with Smad4, translocate into the nucleus, and regulate the transcription of target genes (18). Several small-molecule TβR-I inhibitors have been reported to prevent metastasis of some cancers (19). However, there may be adverse effects of TGF-β inhibition, including potential progression of some cancers because of the repression of TGF-β-mediated growth inhibition of epithelial cells (20).

In this study, we show that administration of the small-molecule TβR-I inhibitor (LY364947) (21) at a low dose, which could minimize the potential side effects of TβR-I inhibitor, can alter the tumor microenvironment and enhance the EPR effect. This effect of low-dose TβR-I inhibitor was demonstrated with two of nanocarriers, i.e., Doxil and a polymeric micelle incorporating ADR (micelle ADR) that we have recently developed (22) [supporting information (SI) Fig. 7]. The present findings strongly suggest that our method, which uses a combination of low-dose small molecule TβR-I inhibitor and long-circulating nanocarriers, is a promising way to treat intractable cancers.

Results

We used the xenografted BxPC3 human pancreatic adenocarcinoma cell line in nude mice as a disease model (Fig. 1). BxPC3 cells do not respond to TGF-β, because of lack of functional Smad4. Hematoxylin/eosin (H&E) staining of tumor tissue in this model (Fig. 1A Left) revealed poorly differentiated histology, with a certain number of blood vessels and thick fibrotic tissue in the interstitium. There was, however, almost no vasculature inside of tumor cell nests (Fig. 1A Right). This model thus represents the histological characteristics of some intractable solid tumors.

Fig. 1.

Histology of BxPC3 xenograft and effects of low-dose TβR-I inhibitor. (A) The histology of the TGF-β-nonresponsive BxPC3 xenograft, used as a model of poorly differentiated pancreatic adenocarcinoma, shown in H&E staining and immunohistochemistry. Examination revealed nests of tumor cells in gland-like structures, with areas rich in fibrotic components (filled by α-smooth muscle actin (SMA)-positive myofibroblasts, shown in red) between them. The tumor tissue also includes some PECAM-1-positive vessels (shown in green) in the interstitium, although almost no vasculature was observed inside the nests of tumor cells. (B) Dextran leakage. At 24 h after administration of low-dose TβR-I inhibitor (1 mg/kg i.p.), i.v.-administered dextran of 2 MDa (50 nm in hydrodynamic diameter) exhibited broader distribution with 1 mg/kg TβR-I inhibitor (Right) than in the control (Left), which was quantified and shown in the graph (n = 12). Error bars in the graphs represent standard errors, and P values were calculated by Student's t test. Ctrl, control; Inhib, inhibitor. (Scale bars, 100 μm.)

Systemic administration of low-dose TβR-I inhibitor in this model significantly altered the characteristic of tumor vasculature at 24 h after administration. We investigated the functional aspects of the effects of low-dose TβR-I inhibitor, using i.v.-administered large-molecule dextran of 2 MDa with a hydrodynamic diameter of 50 nm (23, 24), which is equivalent to the common sizes of nanocarriers (Fig. 1B). Although dextran of this molecular size for the most part remained in the intravascular space in the control condition, as reported in ref. 24, the use of TβR-I inhibitor resulted in a far broader distribution of this macromolecule around the tumor neovasculature. These findings suggest that low-dose TβR-I inhibitor can maintain blood flow in the tumor vasculature and simultaneously induce extravasation of macromolecules.

To investigate the mechanisms of effect of TβR-I inhibitor on the neovasculature, we analyzed the changes in three major components of tumor vasculature, i.e., endothelium, pericytes (Fig. 2), and basement membrane (SI Fig. 8), at 24 h after administration of TβR-I inhibitor. The areas of vascular endothelial cells stained by platelet/endothelial cell adhesion molecule (PECAM)-1 increased slightly with TβR-I inhibitor treatment (Fig. 2B). Although pericyte-coverage of endothelium has been reported to be incomplete in tumors (25), coverage of the endothelium by pericytes, which were determined as NG2-positive perivascular cells, was further decreased by the TβR-I inhibitor treatment. This finding was confirmed by comparing the ratios of PECAM-1/NG2-double-positive areas to PECAM-1-positive areas (Fig. 2C). On the other hand, vascular basement membrane, which was determined by staining with collagen IV, did not differ significantly in the presence or absence of TβR-I inhibitor (SI Fig. 8). We also examined the vasculature in normal organs and found that it was not affected by TβR-I inhibitor in terms of permeability of 2-MDa dextran and morphology on immunostaining (SI Fig. 9).

Fig. 2.

Morphological changes in cancer neovasculature at 24 h after administration of low-dose TβR-I inhibitor. (A) Immunostaining of the tumor neovasculature. NG2-positive pericytes (shown in red) were dissociated (yellow arrows in Right) from VE-cadherin-positive endothelium (shown in green) after TβR-I inhibitor treatment for 24 h. (Scale bars, 50 μm.) (B and C) Areas of PECAM-1-positive endothelium (B) and pericyte-coverage (C) were quantified (n = 40) and are shown in the graphs. Error bars in the graphs represent standard errors, and P values were calculated by Student's t test. Ctrl, control; Inhib, inhibitor.

We next examined the effects of i.p. administration of small-molecule TβR-I inhibitor at a low dose (1 mg/kg) on TGF-β signaling, by determining phosphorylation of Smad2 (SI Figs. 10 and 11). Because it is a small-molecule agent, TβR-I inhibitor transiently suppressed phosphorylation of Smad2. In nucleated blood cells, phosphorylation of Smad2 was significantly suppressed at 1 h after administration of TβR-I inhibitor, but it gradually recovered toward 24 h. In contrast, phosphorylation of Smad2 in tumor cells and most interstitial cells was not suppressed even 1 h after administration, whereas a higher dose (25 mg/kg) of TβR-I inhibitor inhibited Smad2 phosphorylation in most tumor cells. Accordingly, the extent of fibrosis in cancer xenografts treated with low-dose TβR-I inhibitor did not differ from that in the control (SI Fig. 12). On the other hand, low-dose TβR-I inhibitor specifically suppressed the phosphorylation of Smad2 in vascular endothelium (SI Fig. 11B). These findings suggest that the use of small-molecule TβR-I inhibitor at low doses is advantageous for limiting adverse effects.

We thus hypothesized that low-dose TβR-I inhibitor may enhance the accumulation of nanocarriers, the molecular sizes of which are similar to 2-MDa dextran, in hypovascular solid tumors. We used two nanocarriers to test this hypothesis: Doxil (26), a liposomal ADR, and a core–shell type polymeric micelle-encapsulating ADR (micelle ADR) that we developed (22). The latter is a micellar nanocarrier consisted of block copolymers in which ADR is conjugated to the PEG chain through an acid–labile linkage. This drug carrier releases free ADR molecules selectively in acidic conditions, e.g., in intracellular endosomes and lysosomes (SI Fig. 7). We tested the effects of i.p. administration of TβR-I inhibitor with i.v. administration of Doxil or micelle ADR at 8 mg/kg on size-matched xenografts of BxPC3 cells, which are ADR-sensitive in vitro (12). Conventional ADR without drug carriers (free ADR), a small-molecule compound of MW 543.52, was also used for comparison. We first examined the distribution of ADR molecules in tumor tissues by using confocal imaging of fluorescence of ADR and HPLC (Fig. 3). The fluorescence of ADR molecules in micelle ADR is detectable only when ADR molecules are released from the micelle, whereas that in Doxil is detectable even when it is encapsulated in the liposome. The total amount of accumulated ADR, the sum of that in cancer cells and the cancer microenvironment, is measured by HPLC, which detects ADR molecules with and without drug carriers. Administration of TβR-I inhibitor with the nanocarriers yielded significant enhancement of intratumoral accumulation of ADR molecules. Because TβR-I inhibitor did not increase the accumulation of free ADR, we suspected that only macromolecules would be benefited by the use of TβR-I inhibitor through enhancement of EPR effect.

Fig. 3.

Biodistribution of ADR in the BxPC3 model. The biodistribution of ADR was investigated in the BxPC3 model by fluorescence examination (T indicates nests of tumor cells in tumor tissues) and by HPLC. The distributions of Doxil, micelle ADR, and free ADR at 8 mg/kg with and without TβR-I inhibitor at 1 mg/kg were examined 24 h after administration. Enhancement of drug accumulation in tumor was specifically observed with TβR-I inhibitor with Doxil and micelle ADR. Error bars in the graphs represent standard errors, and P values were calculated by Student's t test. Ctrl, control; Inhib, inhibitor.

We then examined the growth-inhibitory effects of these anticancer drugs with and without TβR-I inhibitor on size-matched BxPC3 xenografts. As shown in Fig. 4A, the growth curves of the BxPC3 xenografts confirmed the findings for the distribution of ADR molecules. None of free ADR, Doxil, micelle ADR as monotherapy, or free ADR with TβR-I inhibitor significantly reduced tumor growth. In contrast, ADR encapsulated in nanocarriers exhibited significant effects on the growth of tumor when combined with TβR-I inhibitor (see SI List for statistical study).

Fig. 4.

Effects of TβR-I inhibitor on anti-tumor activity of nanocarriers, incorporating ADR in the BxPC3 model. (A) Free ADR, liposomal ADR (Doxil), micelle ADR (micelle) or vehicle control (ctrl) was administered i.v. in a single bolus with and without TβR-I inhibitor (inhib) i.p. to xenografted mice in which tumors had been allowed to grow for a few weeks before treatment (n = 5). Relative tumor sizes were measured every second day and are shown as a growth curve with bars showing standard errors. Only nanocarriers administered together with TβR-I inhibitor exhibited significant reduction of growth compared with the control. (B) Growth curve study with an increased dose of micelle ADR. With the day of initiation of drug administration designated day 0, anticancer drugs were administered i.v. on days 0, 4, and 8 with and without i.p. TβR-I inhibitor on days 0, 2, 4, 6, and 8. Further growth-inhibitory effect was observed with an increase in dose of micelle ADR. (Results of multivariate ANOVA study are shown in SI List.)

Because micelle ADR was more effective than Doxil (as shown in Figs. 3 and 4A), and the maximum tolerated dose of micelle ADR is far higher than one shot of 8 mg/kg (22, 26) (the dose in Fig. 4A), we further tested the growth-inhibitory effects of an increased dose of micelle ADR combined with TβR-I inhibitor (Fig. 4B). When micelle ADR or free ADR was administered on days 0, 4, and 8, with and without TβR-I inhibitor, only micelle ADR administered together with TβR-I inhibitor exhibited nearly complete growth-inhibitory effect on the tumor in this model. We therefore used this regimen in the following experiments.

The efficacy of combined treatment was further confirmed by using micelle ADR in two other animal models of pancreatic adenocarcinoma. We used size-matched xenograft models of MiaPaCa-2 and Panc-1 cell lines, which are both ADR-sensitive in vitro (12) (Fig. 5 and SI Figs. 13 and 14). MiaPaCa-2 is nonresponsive to TGF-β signaling because of TβR-II deficiency, whereas Panc-1 has no deficiency in TGF-β signaling components and responds to TGF-β. On histological examination, the xenografts of MiaPaCa-2 and Panc-1 exhibited similar undifferentiated pattern with scattered cancer cells, rich fibrous tissue, and sparse vasculature distributed homogeneously, unlike that of BxPC3 xenografts (Fig. 5A and SI Fig. 14A). Use of low-dose TβR-I inhibitor in these models again significantly enhanced the growth-inhibitory effects of micelle ADR (see Fig. 5B, SI Fig. 14B, and SI List for statistical analyses). Effects of free ADR were again not enhanced by TβR-I inhibitor, although the drug itself exhibited some degree of growth-inhibitory effect on the MiaPaCa-2 xenografts. Analysis of the biodistribution of ADR molecules (SI Figs. 13 and 14 C and D) confirmed the effects of TβR-I inhibitor on accumulation of micelle ADR in these cancer models.

Fig. 5.

Growth-curve study in the MiaPaCa-2 pancreatic cancer xenograft model. (A) TGF-β-nonresponsive MiaPaCa-2 cell xenografts exhibited an undifferentiated pattern of histology on H&E staining (Upper), with rich SMA-positive fibrotic tissue (show in red in Lower) and much less PECAM-1-positive vasculature (shown in green) compared with the BxPC3 model. (B) The same experimental protocol as in Fig. 4B was used in the model, and the effectiveness of the use of TβR-I inhibitor was confirmed. Inhib, inhibitor; micelle, micelle-ADR. (Results of multivariate ANOVA for the growth-curve studies are shown in SI List.)

We also tested the growth-inhibitory effect of TβR-I inhibitor and micelle ADR in an orthotopic model of the OCUM-2MLN cell line, which responds to TGF-β (27) (Fig. 6). OCUM-2MLN was derived from a patient with another intractable solid tumor, diffuse-type gastric cancer. The cancer cells were implanted in the gastric wall of nude mice and allowed to grow in situ for 2 weeks, leading to formation of hypovascular and fibrotic tumors in the gastric wall (Fig. 6A). Tumor area (framed by arrowheads in Fig. 6B, Left) was measured before the initiation of drug administration, and tumor growth was evaluated by calculating the relative tumor area at day 16 by measuring tumor area again (Fig. 6B, Right). Significant reduction of tumor growth was again observed only in the mice treated with TβR-I inhibitor and micelle ADR. The distribution of ADR, as detected by fluorescence, confirmed this growth-inhibitory effect (data not shown). These findings suggest that the use of TβR-I inhibitor may enhance the accumulation of nanocarriers in hypovascular solid tumors.

Fig. 6.

Effects of TβR-I inhibitor administered together with micelle ADR in an orthotopic diffuse-type gastric cancer model. OCUM-2MLN, a human diffuse-type gastric cancer cell line, was inoculated into the gastric wall of nude mice (n = 5). Two weeks after inoculation, the cancer tissues exhibited diffuse-type histology on H&E staining (A Upper) with sparse formation of blood vessels (PECAM-1 staining, shown in green) (A Lower). The sizes of tumors on the gastric wall were measured based on tumor areas (B Left), and the values on day 16 were divided by those on day 0, the day of initiation of drug administration, to obtain relative tumor areas. Relative tumor areas are shown with bars for standard errors (B Right). TβR-I inhibitor significantly reduced tumor growth in this model, as well. P values were calculated by Student's t test. Inhib, inhibitor; micelle, micelle-ADR.

Finally, we examined whether low-dose TβR-I inhibitor increases EPR effect specifically in tumor tissues and not in normal organs. Although nanocarriers were originally designed to decrease the drug accumulation in normal organs, it is important to determine whether use of TβR-I inhibitor exacerbates their side effects (SI Fig. 15). In liver, spleen, kidney, blood, and heart, accumulation of ADR as determined by HPLC was not significantly increased by TβR-I inhibitor (SI Fig. 15 A and B). Neither dermatitis nor phlebitis around the tail veins was exacerbated by addition of TβR-I inhibitor (SI Fig. 15C). In addition, the weight of mice that were treated with micelle ADR was not significantly affected by TβR-I inhibitor (data not shown). These findings in normal organs strongly suggest that low-dose TβR-I inhibitor enhances EPR effect only in tumors and that exacerbation of toxicity or side effects of nanocarrier-encapsulated drugs may be minimal with this treatment.

Discussion

In the present study, we have tested a use of TβR-I inhibitor at a low dose to induce alteration in cancer-associated neovasculature to exhibit more leakiness for macromolecules, with less pericyte-coverage and greater endothelial area (Figs. 1 and 2). Because use of TβR-I inhibitor induced the same alteration in neovasculature in the Matrigel plug assay (M.R.K., unpublished data), a model of adult neoangiogenesis (23), the effects of use of TβR-I inhibitor on tumor vasculature observed in the present study may be common in adult neoangiogenesis. Although the roles of growth factors, including TGF-β, may differ during development and in adults, these phenotypes are reminiscent of those of knockout mice deficient in certain components of TGF-β signaling, e.g., endoglin (28, 29), ALK-1 (30, 31), and ALK-5 (32), in which loss of pericyte-coverage and dilatation of the vasculature in yolk sac or embryos were observed. These phenotypes are also consistent with the findings obtained on in vitro culture of endothelial cell lineages (33) and mesenchymal progenitor cells (34), which showed that pericyte maturation is promoted, and endothelial proliferation is inhibited, by TGF-β signaling. Vascular phenotypes due to defects in TGF-β signaling in vivo are also observed in two types of hereditary hemorrhagic telangiectasia (35, 36), which are induced by deficiencies of endoglin or ALK-1, which are components of TGF-β signaling in vascular endothelium. Because of inborn and life-long abnormality of TGF-β signaling in vasculature, these diseases result in a tendency toward hemorrhage in capillaries that is due to vulnerability of the vascular structure. These observations suggest that use of TβR-I inhibitor at a dose corresponding to that in mice in our study may have similar effects in humans. However, the inhibition of TGF-β signaling is only transient in our method, because of the use of small-molecule inhibitor, and the effects of TβR-I inhibitor may thus be far less severe than the phenotypes observed in hereditary hemorrhagic telangiectasia.

The changes in tumor neovasculature induced by TβR-I inhibitor resulted in enhanced extravasation of molecules, although in a molecular-size dependent manner. Accumulation of 2-MDa dextran with a 50-nm hydrodynamic diameter, Doxil with a 108-nm diameter, and micelle ADR with a 65-nm diameter was enhanced by TβR-I inhibitor in the present study, although accumulation of small-molecule agents, including ADR (MW 543.52) and BrdU (MW 307.10) (M.R.K., unpublished data), was not significantly enhanced. Dreher et al. (24) recently reported the molecular-size-dependency of intratumoral drug distribution, using a xenograft model of FaDu cells derived from human hypopharyngeal squamous cell carcinoma. They used several dextrans with molecular sizes ranging from 3.3 kDa to 2 MDa, with estimated hydrodynamic diameters of 3.5 nm to 50 nm, respectively. Dextran molecules of 3.3 kDa and 10 kDa, the smallest ones tested, were found to penetrate deeply and homogeneously into tumor tissue, although they remained in tumor tissue only transiently, for far less than 30 min. However, larger dextran of 2 MDa with a diameter of 50 nm, which we also used in the present study, for the most part remained in the vasculature in cancer tissue and reached only an ≈5-μm distance from the vessel wall at 30 min after injection. Although the histological characteristics of their model, which were not described in their report, may differ from those of the cancer models used in our study, the distribution of 2-MDa dextran observed by Dreher et al. agrees with that obtained without TβR-I inhibitor in the BxPC3 xenografts observed in the present study (Fig. 3). TβR-I inhibitor could thus enhance the accumulation of macromolecules with hydrodynamic diameters of >50 nm, common sizes for nanocarriers, in cancers other than those used in the present study. However, the range of sizes of macromolecules and histological patterns of cancer for which use of TβR-I inhibitor can exhibit enhancing effects remains to be determined.

In conclusion, we have proposed here a use of small-molecule TβR-I inhibitor at a low dose to enhance EPR effect in intractable solid cancers. This method could be a breakthrough in chemotherapy by using nanocarriers in these cancers. Because low-dose TβR-I inhibitor does not affect cancer cells, it may reduce the potential side effects of TGF-β inhibitors, and its enhancing effect is independent of the reactivity of cancer cells to TGF-β signaling. Use of TGF-β inhibitors may thus enable reduction of the systemic doses of nanocarriers and thereby decrease the adverse effects of anticancer drugs.

Methods

TGF-β Inhibitors, Anticancer Drugs, and Antibodies.

TβR-I inhibitor was purchased from Calbiochem (San Diego, CA) (LY364947; catalog no. 616451). ADR was obtained from Nippon Kayaku (Tokyo, Japan) and purchased from Kyowa Hakko (Tokyo, Japan). Doxil was purchased from Alza (Mountain View, CA). Micelle ADR was prepared as reported (22) (see SI Materials and Methods for detailed information). The antibodies to PECAM-1 and VE-cadherin were from BD PharMingen (San Diego, CA), those to neuroglycan 2 and collagen IV were from Chemicon (Temecula, CA), and that to SMA was from Sigma–Aldrich (St. Louis, MO). The anti-phospho-Smad2 antibody was a gift from A. Moustakas and C.-H. Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden).

Cancer Cell Lines and Animals.

BxPC3, MiaPaCa-2, and Panc-1 human pancreatic adenocarcinoma cell lines were obtained from the American Type Culture Collection (Manassas, VA). The OCUM-2MLN human diffuse-type gastric cancer cell line was previously established (27). BxPC3 cells were grown in RPMI medium 1640 supplemented with 10% FBS. MiaPaCa-2, Panc-1, and OCUM-2MLN cells were grown in DMEM with 10% FBS. BALB/c nude mice, 5–6 weeks of age, were obtained from CLEA Japan (Tokyo, Japan), Sankyo Laboratory (Tokyo, Japan), and Charles River Laboratories, (Tokyo, Japan). All animal experimental protocols were performed in accordance with the policies of the Animal Ethics Committee of the University of Tokyo.

Cancer Models.

The effects of anticancer drugs were assessed by s.c. implantation of cancer cells into nude mice, and by orthotopic inoculation of OCUM-2MLN cells into the gastric walls of nude mice. A total of 5 × 10⁶ cells in 100 μl of PBS for the xenograft models and the same number in 50 μl of PBS for the orthotopic model were injected into male nude mice and allowed to grow for 2–3 weeks to reach proliferative phase, before initiation of drug administration. For growth-curve studies, the day of initiation of drug administration was considered day 0, and TβR-I inhibitor, dissolved to 5 mg/ml in DMSO and diluted by 100 μl of PBS, or the vehicle control, was injected i.p. at 1 mg/kg on day 0 only in the experiment shown in Fig. 4A and on days 0, 2, 4, 6, and 8 in other experiments. Doxil, micelle ADR, and free ADR at 8 mg/kg, or normal saline as vehicle control, were also administered i.v. in 200 μl/vol via the tail vein on day 0 (Fig. 4A). In other experiments, micelle ADR at 16 mg/kg, free ADR at 8 mg/kg, or normal saline was also administered i.v. on days 0, 4, and 8. There were five mice per group per cell line. The doses of ADR and Doxil were determined based on the lethal doses in mice (22, 26). For biodistribution studies, three mice per group per cell line were treated with 8 mg/kg Doxil, micelle ADR, or free ADR i.v., with and without TβR-I inhibitor at 1 mg/kg i.p. The mice were examined 24 h after injection.

Quantification in Tumor Models.

Xenograft tumors were measured externally every second day until day 16, and tumor volume was approximated by using the equation vol = (a × b²)/2, where vol is volume, a the length of the major axis, and b is the length of the minor axis. Relative tumor volume was calculated by dividing tumor volume by that on day 0 (the day of initiation of treatment), where actual estimated volumes of xenografted tumors in mm³ at initiation of drug administration were as follows (mean ± standard error): BxPC3 (in Fig. 4A), 76.4± 7.0; BxPC3 (in Fig. 4B), 74.4 ± 3.3; MiaPaCa-2, 221.2 ± 12.7; and Panc-1, 242.16 ± 24.5. For orthotopic OCUM-2MLN tumors, the area of the primary focus on the gastric wall was measured in Adobe Photoshop software, by opening the abdomen before initiation of treatment and at the end of the observation period. Relative tumor area was calculated by dividing tumor area by that on the day of initiation of treatment. The results were further analyzed statistically by the multivariate ANOVA test, using JMP6 software (SAS Institute, Raleigh, NC).

Histology and Immunohistochemistry.

The excised samples were either directly frozen in dry-iced acetone for immunohistochemistry, or fixed overnight in 4% paraformaldehyde and then paraffin-embedded to prepare them for H&E or AZAN staining. Frozen samples were further sectioned at 10-μm thickness in a cryostat, briefly fixed with 10% formalin, and then incubated with primary and secondary antibodies. TOTO-3 for nuclear staining, Alexa488-, Alexa594-, and Alexa647-conjugated secondary antibodies, anti-rat and rabbit IgGs, Zenon labeling kit anti-rabbit and mouse IgG, and FITC-conjugated dextran (MW 2 × 10⁶) were purchased from Invitrogen Molecular Probes (Eugene, OR). Samples were observed by using a Zeiss (Thornwood, NY) LSM510 Meta confocal microscope for immunohistochemistry, and an Olympus (Tokyo, Japan) AX80 microscope for H&E and AZAN staining.

Biodistribution.

Xenografts were inoculated s.c. in nude mice and allowed to grow for 2–3 weeks before drug administration. We then injected TβR-I inhibitor at 1 mg/kg i.p. together with i.v. administration of Doxil, micelle ADR, or free ADR at 8 mg/kg. The tumors or organs were excised 24 h after injection of drugs, and frozen in dry-iced acetone to obtain fluorescence images or weighed and mixed with daunorubicin commensurate with the sample weight as an internal control and then frozen to prepare them for measurement by HPLC. The HPLC method used for analyses is described in ref. 22. To obtain fluorescence images, we performed cryostat sectioning of the frozen samples and washed the sections twice briefly with PBS but did not fix them to avoid elution of ADR. The samples were then observed with a Zeiss confocal microscope, using an excitation laser at 488 nm and a detection filter for the infrared region.

Abbreviations

ADR

adriamycin

EPR

enhanced permeability and retention

PECAM

platelet/endothelial cell adhesion molecule

TβR-I

type I transforming growth factor β receptor.