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Institut de Recerca and
* Department of Surgery of the Hospital de la Santa Creu i Sant Pau, Barcelona; and
Laboratori de Recerca Translacional of the Institut Català d’Oncologia, Barcelona, Spain
1Correspondence: Institut de Recerca, Hospital de Sant Pau, Avda. Sant Antoni M. Claret, 167, Barcelona, Spain. E-mail: rmangues{at}santpau.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: implantation site • carcinoma • metastases
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Moreover, there are differences in growth rate and in response to antitumor drugs between primary tumors and metastases (3
4
5)
and between s.c. and orthotopically implanted human tumor xenografts in nude mice (3
, 6
, 7)
. These findings suggest that changes in cell cycle and/or apoptosis regulation by the organ where the tumor is localized may induce differences in tumor growth and a different predisposition to cell cycle block and/or apoptotic induction by antineoplastic drugs. This hypothesis is based on current knowledge that holds tumor growth to be the net result of cell gain by proliferation and cell loss by apoptosis (8
, 9)
; inhibition of tumor growth by antitumor drugs results from the induction of cell cycle arrest and/or apoptosis in tumor cells (10)
. No study so far has compared the molecular regulation of tumor apoptosis or the cell cycle in tumor cells in xenografts implanted at orthotopic and heterotopic sites. The possible contribution of the organ where the metastasis is located in on its growth has not been studied. Differences in behavior between primary tumor and metastases have traditionally been associated with alteration of growth regulation by the genetic alterations acquired by the primary tumor cells during their progression toward metastases.
To assess the influence of implantation site on tumor growth, we compared alterations in cell cycle and apoptosis of two human pancreatic tumors (NP18 and NP9) implanted in the organ where the tumor grows—pancreas (orthotopically)—or in heterotopic sites, the site of metastases (liver), and two nonmetastatic sites (colon and subcutis). This comparison allowed us to assess the contribution of the implantation site in directing the choice of metastatic site, since direct implantation at the metastatic site eliminates the probable influence of the alterations acquired during metastasis development in this process.
We observed that implantation site changes tumor growth by altering apoptotic induction and the regulation of proteins related to apoptosis or to the cell cycle in a tumor-specific manner. In the NP18 tumor, changes in growth are explained by changes in apoptotic induction and in the activation and/or expression of proteins that regulate or execute apoptosis (AKT, Erk, bcl-XL, Bad, caspase-3, PARP, and FAK). In the NP9 tumor, changes in growth are not explained by apoptotic induction but by changes in the activation and/or expression of the proteins that regulate the cell cycle (Erk, PCNA, and cyclin B1). We also observed that implantation site alters the metastatic pattern of the NP9 tumor, originating a new metastatic site not observed in the orthotopically implanted tumor.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Four-week-old male nu/nu Swiss mice weighing 18–22 g (Iffa-Credo Animaux de Laboratoire, L’Abresle, France) were used for tumor implantation. Animals were housed in a sterile environment; cages, water, and bedding were autoclaved and food was 
-ray sterilized.
Tumor implantation
Tumor fragments (10 mg) of these human pancreatic carcinomas from previous passages were used for implantation at subcutis, liver, colon (heterotopic implantations), or pancreas (orthotopic implantation). Nude mice were anesthetized with 2,2,2-tribromoethanol (Sigma-Aldrich, St. Louis, MO). Implantation was performed depositing the tumor under the subcutis at the right flank of the animals (SC) or sewing it with prolene 6.0 suture at the pancreas (orthotopic, ORT), colon (CL), or liver (LV) as described (11
, 12)
; the incision was closed by stitches. After implantation, mice were inspected twice a week.
Effect of site on tumor growth
Growth was assessed measuring final tumor weight, percentage of macroscopic necrosis, and presence of apoptosis in tumor tissue after death. Important differences in growth for each tumor indicated the need to make two comparisons for each tumor: one that compared growth between ORT and SC sites and another that compared ORT vs. LV and CL. It also required death of the animals to occur at different periods depending on the specific growth rate for each tumor. Thus, mice (n=10) bearing NP9 ORT or SC tumors were killed 4 wk after tumor implantation, whereas NP18 ORT or SC tumor-bearing mice (n=10) were killed 6 wk after implantation. A comparison of tumor growth between ORT, LV, and CL groups (n=5) was performed at 6 wk (for NP9) and 3 months (for NP18) after tumor implantation. At necropsy, final tumor weight and viable tumor tissue weight (tissue left after the macroscopically evident central necrotic core of the tumor was removed) were recorded. Mean tumor weight between groups was compared using the Student’s t test; differences between them were considered significant at a P < 0.05.
Microscopic analysis
Macroscopically viable tumor fragments were frozen in liquid nitrogen or fixed in formalin for molecular or histopathological analyses. After H&E staining of formalin fixed tissue, microscopic morphology of the tumor sections at the different implantation sites was analyzed.
Apoptotic induction
The presence of apoptotic cells within the macroscopically viable tumor tissue was assessed using two independent techniques: Hoechst and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) tests. The Hoechst analysis was performed on formalin-fixed paraffin-embedded tumor tissue. Tissue sections (5 µm) were dewaxed in xylene, dehydrated, and rinsed three times with PBS. Cells were permeabilized, incubated with 0.5% triton X-100 in PBS pH 7.4 for 5 min at room temperature, then rinsed again twice in PBS. Finally, cells were stained with Hoechst (1:5000 in PBS) for 1 h and rinsed with water. Sections were mounted and observed under a fluorescent microscope at 334 nm absorption and 365 nm emission light. Apoptotic induction was studied measuring the formation of DNA strand breaks by TUNEL assay, following the recommendations of the manufacturer (Roche, Nutley, NJ) on formalin-fixed paraffin-embedded tumor tissue.
Analysis of dissemination patterns
The metastatic pattern of each tumor was assessed after ORT, LV, and CL implantation. Animals were killed at 6 wk (NP9) or 3 months (NP18) after implantation. Possible macroscopic dissemination was assessed in most organs of the animal; mediastinum, liver, and lung tissues were collected for inspection of microscopic dissemination. Tissue was fixed in formalin and stained with H&E following the standard protocol.
Western blot analysis
Whole cell protein extracts were prepared from tumor tissue mechanically grounded in lysis buffer, which contains 20 mM Tris/acetate pH 7.5,0.27 M sucrose, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 1 mM Na ortovanadate pH 10.0, 10 mM Na beta-glycerophosphate, 50 mM Na fluoride, 5 mM pyrophosphate, 1% Triton X-100 and Tris acetate-sucrose (20 mM/0.27 M), 0.1% 2-mercaptoethanol, 1 mM benzamidine, 0.2 mM PMSF, 5 mg/ml leupeptin. The amount of protein was quantitated by the Bradford method using the Bio-Rad protein assay dye. Polyacrylamide gels were prepared with stacking (2.7% acrylamide, 0.06% bis-acrylamide, 0.08 M Tris-HCl pH 6.8, 0.1% SDS, 0.1% APS, 0.07% TEMED) and separating (15% acrylamide, 0.003% bis-acrylamide, 0.375 M Tris-HCl pH 8.8, 0.1%SDS, 0.1% APS, 0.07% de TEMED) gels. Samples were denatured at 100°C for 3 min and, after loading 75 µg of total protein, diluted with 3x loading buffer (150 mM Tris-HCl pH 6.8, 6% SDS, 0.15% bromphenol blue, 30% glycerol, 300 mM dithiothreitol), and electrophoresis was run at 30–40 mAmp in Laemmli buffer (25 mM Tris, 250 mM glycine pH 8.3, 0.1% SDS) with molecular weight markers.
After the electrophoresis, samples were transferred at 200 mAmp O/N in transfer buffer (39 mM glycine, 48 mM Tris base, 0.037% SDS, 20% methanol) to nitrocellulose membranes. To control for protein loading, membranes were incubated for 10 min in 0.2% PonceauS (Sigma) in 3% acetic acid and rinsing with water. Membranes were blocked by shaking at room temperature for 1.5 h in TBS-T buffer (132 mM NaCl, 20 mM Tris pH 7.5, 0.1% Tween-20; Sigma) with 5% nonfat milk. Membranes were incubated with the respective primary antibody at the indicated dilution (in TBS-T buffer with 0.1% BSA), shacking for 1 h at room temperature, then with the corresponding secondary antibody. Protein bands were detected by chemiluminescent detection with Supersignal reagent (Pierce, Rockford, IL).
The antibody anti-active MAPK (Promega, Madison, WI) (diluted 1:10,000), which reacts with the doubly phosphorylated forms of Erk-1 and Erk-2, was used to detect the active forms of these two proteins. The rabbit anti-cyclin B1 (1:1000), rabbit anti-Bcl-2 (1:7000 o/n), rabbit anti-Bcl-XL (1:7000 o/n), rabbit anti-FAK (diluted 1:7.000), goad anti-BadP (Ser) (diluted 1:500), goat anti-Bak (1:100), and goat anti-ß-actin (1:15,000) polyclonal Abs were from Santa Cruz Biotech (Santa Cruz, CA. The rabbit anti-cyclin D1 was purchased from Upstate Biotechnology (Lake Placid, NY). The mouse anti-PCNA (1:1000) was from Transduction Laboratories (Becton Dickinson, Rutherford, NJ), the rabbit anti-caspase-3, rabbit anti-caspase-7 and rabbit anti-caspase-8 from PharMingen (San Diego, CA), the rabbit anti-active-Akt from Cell Signaling (New England Biolabs, Beverly, MA), the rabbit anti-PARP from Boehringer Mannheim (Mannheim, Germany), and the mouse anti-p53 was from Calbiochem (San Diego, CA). All secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were used at a dilution of 1:10,000.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). The NP18 tumor had a significantly lower final tumor weight after orthotopic (0.3±0.1 g) than after s.c. (1.2±0.2 g) implantation (P<0.001) (Fig. 1B
). In this tumor, there were no significant differences in final tumor weight between liver and colon implantation. No macroscopic necrosis was observed at any of the implantation sites.
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The NP9 orthotopically implanted tumor reached a significantly higher final weight (12.4±4.3 g) than liver (2.3±0.8 g, P<0.001) or colon (3.4±0.5 g, P<0.001) implanted tumors (Fig. 1A
). Although the NP9 tumor did not show significant differences in final tumor weight between ORT (2.3±0.5 g) and SC (2.2±0.5 g) implantation sites (Fig. 1B
), it showed a significantly (P<0.001) lower level of macroscopic necrosis at the ORT (14.3±4.3%) than at the SC (47.3±9.4%), leading to an increased mass of viable tumor. Liver or colon implantation of this tumor showed no macroscopic necrosis and there were no significant differences in final tumor weight between these two sites.
Microscopic analysis of tumors
When implanted at the colon or liver, NP9 and NP18 tumors altered their morphological appearance by presenting a bigger and whiter cytoplasm compared with ORT and SC sites (Fig. 2
). The NP18 tumor implanted at liver (Fig. 2C
) and colon (Fig. 2D
) presented extensive areas of scar fiber formation (Fig. 2)
, which are generally associated with fibrous tissue growth in the spaces left by extensive cell death. These changes were not detected at the SC (Fig. 2A
) or ORT (Fig. 2B
) sites. These fibers were weakly stained by H&E, indicating they were very young and with low collagen content. Among these fibers, isolated dead tumor cells were observed.
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Differences in tumor cell apoptosis among different implantation sites
We performed nuclear staining with Hoechst dye in NP18 tumor sections implanted at each of the four sites. We observed that within the dense web of fibers in the NP18 implanted at liver (Fig. 2C
) or colon (Fig. 2D
), a significant number of tumor cells could be found that showed apoptotic nuclei (Fig. 3
) whereas no apoptotic cells were detected after ORT (Fig. 3A
) or SC (Fig. 3B
) implantation. The same results were obtained when we measured apoptosis by the TUNEL method. Within the web of fibers in liver (Fig. 3G)
or colon (Fig. 3H
) implanted tumors, numerous tumor cells showed DNA strand breaks (Fig. 3)
, whereas no apoptotic cells were detected after ORT (Fig. 3E
) or SC (Fig. 3F
) implantation. In the NP9 tumor, nuclear staining with Hoechst or the TUNEL assay revealed no apoptotic induction at CL, LV, SC, or ORT implanted tumors.
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Regulation of apoptotic proteins
In NP18 and NP9 tumors there were differences in apoptotic regulation by site, with ORT and SC behaving similarly and differently from CL and LV implanted tumors. Thus, in the NP18 tumor and in the liver and colon implanted tumors, there was a significant increase in caspase 3 activation associated with increased proteolysis of the caspase substrates PARP and FAK, reduced expression of Bcl-XL, activation of AKT, and Bad phosphorylation compared with the orthotopic or s.c. implanted tumors (Fig. 4
A). No changes in Bcl-2 or Bak expression or in proteolysis of caspases 7 or 8 was observed at any of the sites (data not shown). In the NP9 tumor, there was a significant reduction in p53 expression and a significant increase in the expression of the antiapoptotic molecules Bcl-XL and Bcl-2 in liver or colon implanted tumors compared with orthotopic or s.c. implantation (Fig. 4B
). A similarly low level of AKT activation was observed at all four sites; no activation of caspase-3 (Fig. 4B
) or expression of Bak (data not shown) was observed at any of the sites.
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Alteration of cell cycle related molecules
Erk activation in the NP18 tumor was higher for Erk-1 and Erk-2 proteins after SC than after ORT implantation (Fig. 5
A). In this tumor, Erk activation levels after liver or colon implantation were not detectable. In contrast to these differences in Erk activation between sites in the NP18 tumor, PCNA, cyclin D1, or cyclin B1 expression were not regulated, showing similar levels in all four implantation sites (Fig. 5A
). In NP9 tumors, there were differences in cell cycle regulation between sites. ORT and SC implanted tumors behaved equally and differed from liver and colon implanted tumors. Erk activation and PCNA and cyclin B1 expression were significantly lower in liver or colon than in orthotopic or s.c. implanted tumors (Fig. 5B
), whereas cyclin D1 remained at a similar level. We observed a close correlation between activation of Erk and tumor growth rate for both tumors and in all implantation sites (compare Figs. 1
and 5
).
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Pattern of tumor dissemination
When orthotopically implanted, the NP9 tumor gave rise to abundant peritoneal dissemination in all implanted animals. We also observed peritoneal dissemination for heterotopically (liver and colon) implanted NP9 tumors. As expected, no dissemination was recorded after SC implantation. Most important, we detected hepatic metastasis (Fig. 6
A, B) in three of three colon-implanted NP9 tumors and in three of four NP9 liver implanted tumors. Liver metastases were not present in any of the four orthotopically implanted tumors. On the other hand, the NP18 tumor metastasized to the peritoneum and liver when orthotopically implanted. The same metastatic pattern was obtained when this tumor was implanted in the liver or colon. Thus, there were no differences in metastatic pattern among different implantation sites in the NP18 tumor.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Alteration of apoptosis and the cell cycle regulation by tumor implantation site
We first observed that fragments of NP18 and NP9, tumors implanted in liver or colon grew less than those implanted at the ORT or SC site. We hypothesized that this reduced tumor growth would be due to the alteration by the host organ microenvironment of the regulation of the two processes that determine tumor cell growth: cell division and/or cell death. We found a tumor-specific alteration of these processes.
In the NP18 tumor, differences in growth at the different sites were mainly due to changes in apoptotic regulation. Liver and colon implantation demonstrated a lower efficiency than ORT or SC implantation in sustaining the growth of the NP18 tumor. This could be explained by enhanced apoptotic induction, as assessed by the Hoechst and TUNEL assays, concomitant with the induction of scar fiber formation. Apoptosis was associated with activation of executor caspase-3, proteolysis of the caspase substrates PARP and FAK and down-regulation of antiapoptotic proteins. Our results suggest that in this pancreatic carcinoma model, the higher tumor growth occurring at the ORT and SC sites compared with LV and CL is due to a lower level of apoptotic induction, which is accomplished by maintaining the AKT and Erk pathways activated. The activation of these pathways could lead to antiapoptotic signaling through phosphorylation of Bad, release of Bcl-XL, and inhibition of Caspase 3 activation. Bak, Bcl-2, or Caspases 7 and 8 would not be involved in regulating apoptosis in this model. The existence of highly activated AKT and Erk pathways at the ORT site is consistent with the activation of AKT (13
, 14)
and Erks (15)
found in pancreatic adenocarcinomas. Local growth factors (16)
and ECM proteins (17
, 18)
, which are organ specific (19)
, could send signals through AKT, which in turn may phosphorylate Bad (20)
, or through Erk, which also phosphorylates Bad through rsk1 (21
, 22)
. Finally, phosphorylated Bad would be unable to interact with Bcl-XL (23)
and will be free to inhibit the release of Cyt C, caspase activation and apoptotic induction (24
, 25)
. Regulation of Bcl-XL by the Erk pathway has been reported in human pancreatic carcinomas in vitro (26)
.
In contrast, regulation of the cell cycle in the NP18 tumor did not appear to contribute to its differences in growth. Thus, the level of expression of the markers of the different cell cycle phases remained unaltered by site. These included cyclin D1, involved in G1 entry (27
, 28)
, PCNA, required for DNA synthesis in S phase (29)
, and cyclin B1, which regulates the G2-M transition (30)
. This suggests that the regulation of G1 entry after induction of cyclin D1 by Erk activation (18)
is not functional in this tumor and that Erk activation may have a role in driving growth in this tumor not by regulating the cell cycle, but through the antiapoptotic role of Rsk-1 (21
, 22)
.
In the NP9 tumor, alteration of cell cycle regulation rather than apoptosis could explain the observed differences in growth by site. Thus, the lower efficiency of liver and colon sites in sustaining NP9 tumor growth compared with SC or ORT implantation was associated with a significant reduction of the expression of molecules necessary for the passage trough S and G2-M phases. No change in G1 related cyclin D1 expression was detected. In this tumor, the connection between Erk activation and cyclin D1 expression may be disrupted, and alteration of Erk activation most likely changes the cell cycle by altering the expression of PCNA (S phase), and cyclin B1 (G2-M transition). In addition to regulating cyclin D1 expression, Erk has been shown to regulate DNA synthesis and the G2/M transition (31
, 32)
.
Colon or liver implantation of the NP9 tumor did not alter apoptotic induction as measured by the Hoechst staining or TUNEL assays. The NP9 tumor at these sites showed essentially no apoptosis. Moreover, colon and liver implantation did not modify tumor AKT activation, reduced the expression of the p53 protein, did not proteolyze caspase-3, and increased the expression of two antiapoptotic proteins, Bcl-2 and Bcl-XL, with no apparent functional consequence. The lack of apoptotic induction in the NP9 tumor at any implantation site suggests that this tumor has acquired a reduced capacity to enter apoptosis. This is consistent with our finding that the NP9 tumor cultured in vitro was more resistant than the NP18 tumor to induction of apoptosis by several antitumor drugs (5-fluorouracil, farnesyl transferase inhibitor, EGF receptor inhibitor; our own observations). Based on the up-regulation of antiapoptotic proteins Bcl-2 and Bcl-XL, we would expect a reduced sensitivity of tumors implanted at colon or liver compared with SC or ORT sites to stimuli that induce apoptosis, such as exposure to antitumor drugs. We are now testing this hypothesis. The p53 gene is mutated in this tumor; thus, although regulated, it is unlikely to have functional consequences.
Our results suggest that the implantation of any tumor at an heterotopic site where metastases occur (in our case, this would be the liver where the NP18 pancreatic carcinoma metastasizes to) could be used to molecularly analyze the mechanisms contributing to metastatic growth. This is relevant in the light of recent findings suggesting that an important limiting step in the metastatic spread is not the ability to survive in the circulation or extravasation steps, but the ability of the tumor cells to grow at the metastatic site after extravasation (33)
. Study of the molecular differences in signal transduction pathways related to proliferation and apoptosis between the primary tumor and the metastatic sites could provide clues on their differential response to antineoplastic drugs (34
, 35)
. Different sites may differentially regulate pathways that predispose the tumor to different thresholds of apoptosis.
The organ of implantation alters the site of metastases
In our model, implantation at a heterotopic site (colon and liver) determines the acquisition by the NP9 pancreatic carcinoma cells of the capacity to metastasize to a new organ (liver) that was not a target for metastases after orthotopic implantation in nude mice or in the human tumor from which this xenograft was derived. This tumor was derived from a peritoneal metastasis of a human pancreatic carcinoma that metastasized only to the peritoneum. Previous experiments have demonstrated a role for the organ when the tumor is located in the choice of metastatic site. In a human colon carcinoma orthotopic model, Hoffman’s group has shown that lymph node metastases are not generated directly from the primary tumor but by ‘remetastasis’ of hepatic metastases (36
, 37)
, so that competence to colonize the liver determines its ability to metastasize in the regional lymph nodes (38)
. They have also have shown that implantation in serosal layers of nude mouse colon (orthotopic) induces local invasion and lymph node metastases, whereas implantation in the serosal layers of the stomach (heterotopic site) limits invasiveness and represses this metastatic capacity (39)
.
Our findings are novel in that colon and liver implantation sites conferred information to the pancreatic tumor cells so that they acquire a new metastatic capacity, which was not observed in the patient. This finding suggests that not only the interaction with the organ where tumor cells arrive, but their previous interaction with other organs may determine the final fate of the metastases. Our findings also indicate that in our model, growth and metastases are independent processes regulated by different molecular pathways. The interaction of tumor cells with the liver or colon confers on them a slower growth rate as well as new metastatic potential. The organ with which the tumor interacts through epigenetic mechanisms appears to be as important as the genetic mutations the tumor bears in determining tumor functions. Although genetic mutations confer anchorage-independent growth, which correlates with tumorigenicity, this does not mean that the tumor cell is completely autonomous in growth, but needs interaction with the organ where it stands to express functions that further its progression, which may be the regulation of the cell cycle or apoptosis or the predisposition to future metastatic spread.
In summary, we observed that implantation site changes tumor growth by altering apoptotic induction and the regulation of proteins related to apoptosis or to the cell cycle in a tumor-specific manner. In the NP18 tumor, changes in growth are explained primarily by changes in apoptotic induction, through AKT and Erk regulation and activation of the Bad/Bcl-XL/caspase-3 pathway. In the NP9 tumor, changes in growth are not explained by apoptotic induction but by changes in the activation and/or expression of the proteins that regulate the cell cycle (Erk, PCNA, and cyclin B1). We also observed that implantation site alters the metastatic pattern of the NP9 tumor, originating a new metastatic site not observed in the orthotopically implanted tumor. Our findings may hold relevance in the pursuit of better animal models for tumor progression and their use to evaluate antitumor activity against primary tumors and metastases.
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ACKNOWLEDGMENTS |
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Received for publication December 3, 2001. Accepted for publication March 6, 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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