To develop and evaluate new therapeutic strategies for the treatment of human cancers, well-characterised preclinical model systems are a prerequisite. To this aim, we have established xenotransplantation mouse models and corresponding cell cultures from surgically obtained secondary human liver tumours. Established xenograft tumours were patho- and immunohistologically characterised, and expression levels of cancer-relevant genes were quantified in paired original and xenograft tumours and the derivative cell cultures applying RT-PCR-based array technology. Most of the characteristic morphological and immunohistochemical features of the original tumours were shown to be maintained. No differences were found concerning expression of genes involved in cell cycle regulation and oncogenesis. Interestingly, cytokine and matrix metalloproteinase encoding genes appeared to be expressed differentially. Thus, the established models are closely reflecting pathohistological and molecular characteristics of the selected human tumours and may therefore provide useful tools for preclinical analyses of new antitumour strategies in vivo.
The liver is a common site of
distant metastasis originating from different neoplasms including
gastrointestinal (pancreatic, stomach, colorectal), lung and breast cancers.
Also primary liver tumours such as cholangiocellular carcinomas (CCC), cancers
of the bile ducts [
Due to its poor prognosis and unsatisfying
treatment options, suitable animal models for secondary liver cancer are
required as a prerequisite for studying factors involved in the pathogenesis of
the disease as well as for the development and evaluation of new anticancer
therapies. Various approaches include the use of transgenic or knockout mice
[
Thus, in the present study liver metastases derived from various human adenocarcinomas were used to establish subcutaneous xenograft tumours in SCID/beige mice. Extensive histological analyses were performed to demonstrate that the transplants widely reflect the characteristics of the parental lesion. In addition, gene expression profiling by means of RT-PCR-based microarrays revealed that expression of cancer-related genes appeared to be similar in corresponding original and xenograft tumours as well as in derived cell cultures. Therefore, we conclude that the established tumour models and cell cultures may represent valuable tools for the development and analysis of new treatments targeting secondary liver tumours.
Primary
and secondary liver tumours were obtained from patients at the time of liver
transplantation or surgical resection of the neoplasm. Immediately after
surgical resection, tumour samples were transferred into transport medium (RPMI 1640, Sigma-Aldrich, Wien, Austria) containing 10%
heat-inactivated foetal bovine serum (FBS) (PAA, Pasching,
Austria), 100 U/mL penicillin-streptomycin (PAA), 2.5
Human
tumour samples with an average size of 1 cm3 were cut into 2 × 2 mm
pieces in the presence of digestion medium (PBS/2 mg/mL collagenase III; 37°C; Worthington
Biochemical Corporation, NJ,
USA), transferred into 15 mL tubes (Sarstedt, Wiener Neustadt, Austria) and further incubated for 1 hour at 37°C with
continuous shaking at 320 cycles per minute (Thermoshaker HTMR 132; Haep Labor Consult, Bovenden,
Germany). To stop
digestion, an equal volume of culture medium (DMEM/10% FBS/50 U/mL penicillin-streptomycin)
was added. The obtained single cell suspension was centrifuged at 180 xg for 5
minutes, and cells were washed twice with PBS. The cell pellet was resuspended
in 1 mL of injection medium (RPMI 1640 phenol red free/1%
penicillin-streptomycin) and 150-200
Tumour
pieces either obtained from primary (AKH23, KFJ18) or xenografted tumours
(AKH10, KFJ6, KFJ9, KFJ10) were processed as described above, and obtained
single cell suspensions were transferred into cell culture flasks (Sarstedt)
containing culture medium. Established
cell cultures were characterised by immunocytochemistry using antibodies
reacting with human and mouse major histocompatibility complex (MHC) class I
antigens. Briefly, cells were incubated with a R-phycoerythrin—conjugated mouse
anti-human leukocyte antigen (HLA)-A,B,C (BD Pharmingen, Schwechat, Austria) or a fluorescein isothiocyanate
(FITC)—conjugated mouse
antimouse H-2Dd monoclonal antibody (Becton Dickinson, Heidelberg, Germany) for one hour at 4°C in the dark. Cells
were washed twice, resuspended in PBS, and subjected to FACS analysis
(FACScalibur, Becton Dickinson). In addition, cells were stained with an
antibody directed against a human epithelial-specific antigen (ESA; Serotec, Düsseldorf, Germany) followed by detection with FITC-conjugated
polyclonal rabbit anti-mouse immunoglobulin (DakoCytomation, Glostrup, Denmark). After
characterisation, cells usually with passage numbers 5–10 were frozen in
liquid nitrogen for long-term storage. On demand cells were thawed and expanded
for further in vitro analysis
or retransplantation into immunodeficient mice. Therefore,
Xenograft
tumours after the first or second passage in mice were excised and fixed in 4%
buffered formalin (pH 7.0, Sigma-Aldrich) and embedded in paraffin (Histo-Comp,
Sanova, Wien, Austria)
using automatic embedding equipment (Tissue Tek, Miles Scientific, Inc., Ill, USA). Three
RNA was extracted from
trypsinised cells or frozen and pulverised tumour samples according to the
RNeasy Mini Kit protocol (Qiagen, Wien,
Austria) and treated afterwards
with Turbo DNase (Ambion, Tex,
USA) according to the manufacturer’s instructions. Subsequently, 150 ng
of total RNA were reverse transcribed using the iScript cDNA synthesis Kit (Bio-Rad
Laboratories, Calif, USA). 50
Sequences amplified on TaqMan low density arrays.
Gene | Gene name | Classification | TaqMan assay IDa |
---|---|---|---|
BCL2 | B-cell CLL/lymphoma 2 | Inhibition of apoptosis | Hs00153350_m1 |
CCND1 | Cyclin D1 | Kinase activator, cell cycle control, proliferation | Hs00277039_m1 |
CDC25B | Cell division cycle 25B | Protein phosphatase, cell proliferation | Hs00244740_m1 |
CDKN1B | Cyclin-dependent kinase inhibitor 1B (p27, Kip1) | Cell cycle control, tumour suppressor | Hs00153277_m1 |
CTNNB1 | Catenin (cadherin-associated protein), beta 1, 88 kDa | Cytoskeletal protein, cell adhesion, oncogenesis | Hs00170025_m1 |
EGFR | Epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog) | Cell cycle control, proliferation, oncogenesis | Hs00193306_m1 |
ERBB2 | v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 | Protein kinase receptor, oncogenesis, cell cycle control | Hs00170433_m1 |
ETV4 | ets variant gene 4 (E1A enhancer binding protein, E1AF) | Transcription factor, oncogenesis, cell motility | Hs00385910_m1 |
IL6 | Interleukin 6 (interferon, beta 2) | Chemokine, inhibition of apoptosis | Hs00174131_m1 |
IL6R | Interleukin 6 receptor | Cell proliferation, immunity and defense | Hs00169842_m1 |
IL8 | Interleukin 8 | Angiogenesis, cell proliferation/differentiation | Hs00174103_m1 |
KRAS | v-Ki-ras2 Kirsten rat sarcoma 2 viral oncogene homolog | Small GTPase, cell proliferation/differentiation | Hs00270666_m1 |
MET | Met proto-oncogene (hepatocyte growth factor receptor) | Protein kinase receptor, oncogenesis | Hs00179845_m1 |
MMP1 | Matrix metalloproteinase 1 (interstitial collagenase) | Metalloprotease, extracellular matrix break down | Hs00233958_m1 |
MMP11 | Matrix metalloproteinase 11 (stromelysin 3) | Metalloprotease, inhibition of apoptosis | Hs00171829_m1 |
MYC | v-myc myelocytomatosis viral oncogene homolog | Oncogene, cell cycle control | Hs00153408_m1 |
PTGS2 | Prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase, Cox-2) | Oxidoreductase, lipid metabolism, deregulated in epithelial tumours | Hs00153133_m1 |
SERPINB5 | Serine proteinase inhibitor, clade B (ovalbumin), member 5 | Proteinase inhibitor, oncogenesis | Hs00184728_m1 |
VEGFA | Vascular endothelial growth factor | Growth factor, angiogenesis | Hs00173626_m1 |
VEGFC | Vascular endothelial growth factor C | Cell proliferation and differentiation | Hs00153458_m1 |
WNT1 | Wingless-type MMTV integration site family, member 1 | Signalling molecule, oncogenesis | Hs00180529_m1 |
rRNA18Sb | 18S ribosomal RNA | Eukaryotic ribosomal RNA gene, obligatory control | 4342379-18S |
GUSBb | Glucuronidase, beta | Galactosidase, carbohydrate metabolism | Hs99999908_m1 |
ACTBb | Actin, beta | Cytoskeletal protein | Hs99999903_m1 |
am1 indicates that the TaqMan minor groove binding probe spans an exon junction and will not detect genomic DNA;
bendogenous control genes shown in a pilot study (TaqMan human endogenous control plate) to be equally expressed in all samples investigated.
To
identify genes expressed differentially in all xenografts and parental tumours
analysed, a Wilcoxon paired-samples test was performed (SPSS for Windows Vs. 11.5). Statistical
significance was defined as
Human secondary
liver tumour tissue was obtained from patients at the time of surgery or
resection of the neoplasm. In total, tumour samples from 17 patients including liver
metastases of colorectal carcinomas (
Relevant characteristics of original human tumour samples.
Tumour ID | Age | Sex | Diagnosisa |
---|---|---|---|
AKH10 | 72 | m | Intrahepatic multifocal CCC |
AKH23 | 65 | f | Pancreatic adenocarcinoma derived liver metastasis |
AKH47 | 63 | m | Intrahepatic CCC |
KFJ6 | 75 | m | CRC derived liver metastasis |
KFJ9 | 55 | f | Intrahepatic metastatic CCC |
KFJ10 | 65 | f | CRC derived liver metastasis |
KFJ12 | 78 | m | CRC derived liver metastasis |
KFJ18 | 64 | f | CRC derived liver metastasis |
KFJ21 | 52 | m | CRC derived liver metastasis |
KFJ25 | 73 | m | CRC derived liver metastasis |
aCCC: cholangiocellular carcinoma; CRC: colorectal carcinoma.
In order to compare original and
xenograft tumours morphologically, sections were stained with haematoxylin/eosin
and examined by light microscopy. Representatively for colorectal liver
metastases, sections of the original tumour KFJ6 and its derived xenograft are
shown (see Figures
To further
characterise the established xenograft tumours and their corresponding original
counterparts, immunohistological stainings for detection of CEA were performed.
CEA is a glycoprotein expressed in adenocarcinomas of the intestinal tract and
in other tumours of epithelial origin such as lung adenocarcinoma, pancreatic
adenocarcinoma, and cholangiocellular carcinomas (CCCs) [
Immunohistochemical analyses of human original and corresponding xenograft tumours. CCC: cholangiocellular carcinoma; CRC: colorectal carcinoma; n.a.: not analysed; −: no staining; +: positive staining; bsingle stained cells or staining restricted to normal liver cells; orig.: original tumour sample; xeno.: xenograft tumour sample.
Tumour | Diagnosis | CEA orig./xeno. | CK8/18 orig./xeno. | CK20 orig./xeno. |
---|---|---|---|---|
AKH10 | Intrahepatic metastatic CCC | +/+ | +/+ | −/− |
AKH23 | Liver metastasis of pancreatic cancer | +b/− | +/+ | −/− |
AKH47 | Intrahepatic CCC | +b/− | +/+ | −/− |
KFJ6 | CRC liver metastasis | +/+ | +/+ | +/+ |
KFJ9 | Intrahepatic metastatic CCC | +/+ | +/+ | +/+ |
KFJ10 | CRC liver metastasis | +/+ | +/+ | +/+ |
KFJ12 | CRC liver metastasis | +/+ | +/+ | +/+ |
KFJ18 | CRC liver metastasis | +/+ | +/+ | +/+ |
KFJ21 | CRC liver metastasis | +/+ | +/+ | +/+ |
KFJ25 | CRC liver metastasis | +/+ | +/+ | +/+ |
In order to compare the established xenograft
tumour models with the respective original tumour counterparts on a molecular
basis, gene expression analyses were performed. For this purpose, relative
expression levels of a number of cancer-relevant genes (see Table
Relative differences in gene expression levels (n-fold) of
original tumour samples compared to the corresponding xenograft
tumour. Indicated
values represent the mean of three measurements including the calculated
standard deviation. Ratios were calculated after normalisation of individual
RNA amounts to a standard reference RNA. Values indicating differences higher
than 2.5-fold are printed in bold. Gene symbols correspond with Table
AKH10 | AKH23 | KFJ6 | KFJ9 | KFJ10 | KFJ12 | KFJ18 | KFJ21 | |
---|---|---|---|---|---|---|---|---|
BCL2 | ||||||||
CCND1 | ||||||||
CDC25B | ||||||||
CDKN1B | ||||||||
CTNNB1 | ||||||||
EGFR | ||||||||
ERBB2 | ||||||||
ETV4 | ||||||||
IL6 | n.d.# | n.d.# | n.d.# | n.d.# | n.d.# | |||
IL6R | n.d.# | |||||||
IL8 | ||||||||
KRAS2 | ||||||||
MET | ||||||||
MMP1 | n.d.# | n.d. | n.d. | n.d.# | ||||
MMP11 | n.d.# | |||||||
MYC | ||||||||
PTGS2 | n.d.# | n.d.# | n.d.# | n.d.# | n.d.# | n.d.# | ||
SERPINB5 | n.d. | |||||||
VEGFA | ||||||||
VEGFC | n.d.# | n.d.# | n.d.# | n.d.# | n.d.# | n.d.# | n.d.# | |
WNT1 | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. |
Finally, gene expression
levels of original and xenograft tumour samples exemplarily were compared to those
of their derived cell cultures (see Table
Relative differences in gene expression levels (n-fold) within tumour samples compared to derived cell cultures. n.d.: not determinable, Ct values obtained with cDNA derived either from cultured cells ($) or from both tumour samples and cells were below threshold (>39).
AKH23 original cells | KFJ9 xenograft cells | KFJ10 xenograft cells | |
---|---|---|---|
BCL2 | |||
CCND1 | |||
CDC25B | |||
CDKN1B | |||
CTNNB1 | |||
EGFR | |||
ERBB2 | |||
ETV4 | |||
IL6 | n.d.$ | n.d.$ | |
IL6R | |||
IL8 | |||
KRAS2 | |||
MET | |||
MMP1 | n.d. | ||
MMP11 | n.d.$ | ||
MYC | |||
PTGS2 | n.d. | n.d. | |
SERPINB5 | |||
VEGFA | |||
VEGFC | n.d.$ | n.d. | n.d. |
WNT1 | n.d. | n.d. | n.d. |
Tumour mouse models as well as tumour-derived cell lines are a prerequisite for the development and evaluation of new and existing tumour therapies. Although a number of xenograft models have been published for colorectal carcinomas and pancreatic adenocarcinomas in most cases, these were established from cultured cell lines available for example from ATCC. In these examples, it is not clear how long-term cultivation of these (mostly poorly characterised) cells affects tumour formation and biology. Therefore, we decided to establish xenografts directly from patient tumours and subsequently analyse both tissues in detail to demonstrate that the generated model closely reflects the original malignancy. In the present study, we report the establishment and detailed characterisation of human xenograft tumour models derived from secondary liver cancer, that is, tumour metastases originating from colorectal, cholangiocellular, and pancreatic cancers. Xenografts were established directly from tumour biopsies omitting culturing of isolated cells, which may cause development of tumours that do not share the characteristics of the respective original due to the selection and expansion of specific cell clones. The applied method of enzymatic digestion of whole tumour samples followed by injection of a mixture of tumour and stromal cells was shown to overcome this obstacle. With respect to xenografts derived from colorectal carcinomas, the applied method resulted in a take rate of 60% and 50%, respectively, when cholangiocellular carcinoma-derived cells were injected. Retrospective analysis of xenograft tumour growth with clinical data of the respective patient did not reveal any significant correlation. Instead, the condition of the primary tumour sample, for example, the presence of large necrotic areas appeared to be critical.
Pathohistological examination of the established xenografts and comparison to their respective original tumours demonstrated that the typical morphology of the tumours was retained after xenotransplantation. Moreover, immunohistological analyses showed that each of the established xenograft tumours retained the typical tumour-specific antigen profile observed in the original tumour sample. Cell cultures established either from original or xenograft tumour tissues were shown to be of epithelial origin and not contaminated with murine cells (data not shown). Although the respective tumour transplants could be passaged in mice for extended periods (up to 30 times) without major changes in growth behaviour and morphology (data not shown), a cryoconservation protocol was established facilitating storage of samples at early passages to avoid development of histopathological alterations over time. Retransplantation experiments with tumour samples frozen for different time spans (3, 6, and 12 months) revealed an average take rate of 70% to 100% in both SCID/beige and nude mice.
Molecular
characterisation based on quantitative gene expression analyses using human
specific primers and probes revealed that in most of the corresponding original
and xenograft tumour samples expression of oncogenes and genes involved in cell
cycle regulation appeared not to be affected by the xenografting process. Major
differences within original and xenograft tumour samples as well as their
derived cell cultures were detected regarding genes encoding cytokines (IL-8,
IL-6) and matrix metalloproteinases (MMP-1, MMP-11). This finding can be
explained by the fact that these molecules are rather expressed by inflammatory
cells (monocytes, neutrophils), stromal fibroblasts, and endothelial cells than
by the tumour cells themselves. A high level IL-8 expression, however, was also
reported in cultured colon carcinoma cells, where it was associated with the
metastatic behaviour of these cells [
Matrix
metalloproteinases (MMPs) are a family of extracellular matrix degrading
enzymes, which have their physiological role in tissue remodelling processes
such as embryonic development or wound healing [
The developed carefully characterised
human xenograft tumours derived from secondary liver tumours share assertive
characteristics with their respective original human counterparts. In addition,
the established cell cultures offer the possibility to evaluate new therapeutic
strategies in vitro before
their use in vivo in the
corresponding tumour mouse models. These valuable tools might be used for the
development and preclinical evaluation of new therapeutic drugs as well as of
alternative methods such as expression targeted retroviral vectors [
The authors thank Bettina Grasl-Kraupp and Hannes Zwickl, Institute of Cancer Research, Medical University Vienna, and Stefan Stättner, Department of Surgery, Kaiser-Franz-Josef-Spital Vienna, for providing primary human liver tumour tissue. They also thank Michaela Wendl and Marielle König-Schuster for taking care of the animals. In addition, they acknowledge the excellent technical assistance of Doris Rosenfellner and the support and technical advice of Ingrid Walter, both at the University of Veterinary Medicine, Institute of Histology and Embryology. The authors also appreciate the help of Irene Sommerfeld-Stur, Institute of Animal Breeding and Genetics, in statistical analysis of the presented data. This work was funded by the Austrian Genome Research Program GEN-AU GZ200.058/6-VI/2/2002. The work of M. Stürzl was funded by a grant provided from the Interdisciplinary Center for Clinical Research (IZKF) of the University of Erlangen-Nürnberg.