Background
Cancer research has received much attention and funding over the past decades, reflecting its increased incidence and significance as a public health problem. Carcinogenesis is a multifaceted and complex disease process, making malignancies inherently difficult to treat, while at the same time presenting multiple pathways for investigation as management options. Novel treatments targeting these different pathways can then be assessed, although tumours in the brain have been excluded from many clinical trials due to the restrictive nature of the blood–brain barrier (BBB), often making brain metastases not accessible to novel treatments [
1,
2]. Metastatic brain tumours are present in 22-30% of patients diagnosed with breast cancer [
3‐
5], therefore making animal models of brain metastases important tools to explore adequate treatment options for this aspect of the disease.
The process of brain metastases involves cells from a primary tumour entering blood vessels, avoiding death signals in the circulation, then undergoing extravasation through the BBB [
6]. The BBB is a dynamic interface between the cerebral circulation and brain tissue, and acts to protect the brain microenvironment [
7]. While investigating metastases, many scientists using cell culture presume that tumour cell lines will behave indefinitely in a uniform manner, although several studies have demonstrated that this is not the case. Changes exhibited with extended
in vitro growth time, high passage number and cross contamination with other cell lines have been frequently described in the literature [
8‐
11], particularly when cancer cell lines are obtained from sources other than reputable major cell libraries [
12]. There is the assumption that well characterised cell lines available from cancer cell repositories are verified and maintained at a high standard, meaning that researchers do not need to authenticate these cell lines before commencing their experiments [
13]. In the current study we report differential characteristics of the same cancer cell line obtained from two different reputable cell banks, suggesting that researchers cannot assume that cells obtained from reputable cancer cell repositories will all behave identically.
Discussion
In the current study, Walker 256 cells obtained from the CRCTU had potent tumorigenic properties when compared to the ATCC Walker 256 breast carcinoma cells. Evidence of this includes the substantially increased incidence of tumour growth and tumour volume after CRCTU Walker 256 inoculation in the two tumour models used in this study, as well as the fact that only CRCTU Walker 256 internal carotid artery injected animals developed tumours in the eye, temporalis muscle and lung. It has been shown in previous studies that different tumour cell lines cloned from the same neoplasm may have different tumorigenic properties when implanted
in vivo[
14,
15]. However, cell lines developed from a single mouse mammary tumour that showed differing culture morphology and growth characteristics
in vitro, resulted in tumours that displayed similar histology to each other and comparable tumorigenicity when injected into syngeneic hosts [
16].
Despite the fact that both populations of Walker 256 breast carcinoma cells were obtained from reputable tumour cell banks that described the Walker 256 cell line as tumorigenic in Wistar rats, there was considerable variability in their genetic profile and subsequently growth behaviour
in vivo and morphology
in vitro. This was despite the fact that both Walker 256 cell lines used in this study were shown to be of rat origin with no evidence of contamination by other mammalian cell lines. ATCC has been instrumental in the push to develop a standard method of cell line verification involving short tandem repeat profiling along with the development of a database of short tandem repeat profiles for commonly used cell lines [
17,
18].
Control of cancer cell tumorigenicity has been extensively studied, predominantly in relation to genetic control of cancer growth
in vivo. For example, p75 has been linked to reduced neuroblastoma tumorigenicity [
19]. However, characteristics of tumour cells in culture have also been investigated, with shorter doubling time, reduced monolayer density, poor motility and lower incidence of focus formation
in vitro linked to decreased tumorigenicity of cell lines when used
in vivo[
20,
21], although these experiments were generally comparing different cell lines. In contrast, the current study aimed to determine the differences between the same cell line obtained from two different sources.
The CRCTU Walker 256 breast carcinoma cells, found to be more tumorigenic than their ATCC counterparts, showed darker nuclear staining and increased nucleus to cytoplasm ratio when compared to the flatter more eosinophilic ATCC Walker 256 cells. There have been few previous studies to determine the relationship between cell morphology and cancer cell tumorigenicity. Further investigation is required to determine if the characteristics observed in this experiment are related to the tumorigenicity of the cells described. Furthermore, previous studies have suggested that behaviour of cancer cell lines
in vitro is poorly correlated with tumorigenicity
in vivo[
21]. Despite this, in the current study morphological features seen
in vitro for Walker 256 cells from both the CRCTU and ATCC were closely associated with the morphology evident
in vivo.
There are many plausible explanations for the differential characteristics evident for CRCTU and ATCC Walker 256 breast carcinoma cells in this study. It is possible that variations in storage methods, extended culture times and high passage number may have contributed to the differences seen in the same cell line obtained from the CRCTU and the ATCC. Immortalised tumour cell lines evolve over time in animal models where malignancies are induced by inoculation with a homogenous population of tumour cells [
22]. Conversely, human neoplastic tissue is not a uniform entity. Within a tumour mass, there exist various heterogeneous subpopulations of tumour cells with different metastatic potential and diverse propensity to metastasise to various organs [
23,
24].
Tumour cells harvested from a neoplasm
in vivo have been known to develop characteristics over time
in vitro that are distinct from those evident in the original cancerous tissue [
14]. The proposed reason for this phenotypic change is that more aggressive or mitotic properties are favoured by clonal selection
in vitro, with highly metastatic varieties more phenotypically stable [
25,
26]. Long term passage of Walker 256 cells has previously been shown to alter chemotactic behaviour
in vitro[
27].
Walker 256 carcinoma is rat mammary tumour cell line that originally occurred spontaneously in a pregnant albino Sprague-Dawley rat [
28]. The Walker 256 cell line has been used previously to establish experimental brain metastases through an internal carotid artery injection and direct implantation into the cerebral cortex [
29‐
33].
Tumour growth evident following both inoculation methods of CRCTU Walker 256 cells showed larger tumour volume in a shorter period of time, when compared to previous experiments described in the literature using the Walker 256 cell line, although the incidence was comparable [
34‐
37]. In contrast, the ATCC Walker 256 cells showed a much lower incidence and longer incubation period required to form only a single tumour when compared to these previous studies. Therefore, neither the CRCTU, nor the ATCC Walker 256 breast carcinoma cells behaved exactly as previous studies have described, although the CRCTU population were more analogous to the literature.
Despite the consistency of the direct injection model of tumour induction, the ATCC Walker 256 cells did not grow any tumours through the use of this method. Thus the extravasation process through the BBB is not the limiting factor for ATCC Walker 256 tumour growth in the brain. Furthermore, 11% of animals grew metastatic brain tumours 10 weeks following ATCC Walker 256 inoculation into the internal carotid artery, meaning that at least some of the tumour cells were able to complete the extravasation process.
The CRCTU Walker 256 inoculated animals for both the internal carotid artery and the direct inoculation model showed a significant increase in albumin immunoreactivity when compared to the culture medium group. It is likely that albumin immunoreactivity was increased in response to the substantial tumour growth evident in the CRCTU Walker 256 tumour inoculated groups and subsequent increased BBB permeability. It is well accepted in the literature that blood vessels within brain metastases of breast cancer are more permeable than BBB microvessels, as they are characteristic of the breast tissue origin of the tumour cells causing substantial cerebral oedema [
29,
38‐
41]. Furthermore, it has been postulated that the permeability of blood vessels surrounding brain metastases is also increased, which may explain the widespread albumin immunoreactivity evident 9 days following CRCTU tumour injection into the internal carotid artery.
The ATCC tumour cell inoculated animals only grew one tumour in either model of metastatic brain tumour induction, which was not sufficient to cause a significant difference in albumin immunoreactivity from vehicle level and thus did not increase the permeability of the BBB. This shows that the presence of tumour cells with low tumorigenicity in the brain microcirculation do not cause an inflammatory reaction disrupts the normal function of the BBB. Furthermore, ATCC Walker 256 localisation in the neuropil of the striatum did not cause long term damage to the brain sufficient to increase the permeability of the BBB 4 weeks following direct injection.
A rim of reactive glial cells is often evident surrounding metastatic brain tumours in human surgical tissue [
42], as was also apparent surrounding tumours grown in this study. The pattern of glial cell reaction was different surrounding CRCTU and ATCC Walker 256 tumours that grew following internal carotid artery inoculation. The location of CRCTU tumours within the lateral ventricles may be the cause of these differences, as the mass is in less direct contact with the neuropil. In contrast, the single tumour that grew 10 weeks following internal carotid artery inoculation of ATCC Walker 256 cells, showed much more extensive microglial infiltration along with increased microglia and astrocytes surrounding the tumour. A proposed function of this glial halo is to act as a barrier to the flow of oedematous fluid [
43]. Astrocytes and microglia may proliferate and become activated in response to contact with serum proteins, such as albumin which is present in oedematous fluid that accumulates around the tumour [
33,
44]. However it is also possible that in the direct inoculation model the glial reaction could be caused in part by reaction to needle track injury, particularly for the animals that were euthanized 7 days following CRCTU Walker 256 inoculation.
The low tumorigenicity of ATCC Walker 256 cells may be the reason that these cells did not show the same influence on the brain microenvironment as CRCTU Walker 256 growth. This is demonstrated by the significant increase in IBA1 and GFAP labelled cells following both internal carotid artery and direct injection of CRCTU Walker 256 tumour cells when compared to the culture medium injected groups. However this phenomenon was not evident following ATCC Walker 256 tumour inoculation for either model used in this study. Thus, the presence of low tumorigenicity cancer cells in the brain microcirculation or the neuropil, did not show significant interaction with the host microenvironment.
Methods
Cell culture
Walker 256 breast tumour cells (rat) were obtained from two cell banks, the American Type Culture Collection (ATCC), and the Cell Resource Centre for Medical Research at Tohoku University (CRCTU). The Walker 256 cells obtained from the ATCC were reported to be passage number 290. However, the CRCTU did not provide details of passage number for the Walker 256 cells. These two cell populations were cultured for a maximum of 30 additional passages, in the same incubator. Both the Walker 256 cell populations were cultured according to the instructions from the respective cell bank. Briefly, Walker 256 cells from ATCC were cultured in growth medium consisting of Sigma 199 M4530 culture medium containing 5% sterile normal horse serum and 1 mL of penicillin and streptomycin (Sigma 10,000 units of penicillin and 10 mg of streptomycin/mL) for each 100 mL volume, while Walker 256 cells from the CRCTU were cultured in growth medium made up of Sigma RPMI-1640 culture medium containing 10% sterile foetal bovine serum and 1 mL of penicillin and streptomycin (Sigma 10,000 units penicillin and 10 mg of streptomycin/mL) for each 100 mL volume.
Culture flasks of 150 cm2 were used to grow the cells and once >90% confluence was reached, the cells were detached by the addition of 3.5 mL of 1% trypsin (Sigma) or 3.5 mL of 0.02% EDTA for ATCC and CRCTU Walker 256 cells, respectively. The cells were spun down in a centrifuge (5 minutes at 1500 RPM) and then resuspended in serum free culture medium. The number of cells was calculated using a haemocytometer and then diluted, so that there was between 105 and 106 cells in every 0.2 mL of cell suspension for internal carotid artery injection, or the same number of cells in 8 μL for direct inoculation into the brain.
Animals
The experimental procedures described throughout this project were performed within the National Health and Medical Research Council (NHMRC) guidelines and were approved by both the University of Adelaide and Institute of Medical and Veterinary Science Animal Ethics Committees. All experiments complied with the EC Directive 86/609/EEC for animal experiments. Male Wistar rats weighing 250-350 g were group housed in the IMVS Animal Facility and were supplied with a diet of rodent pellets and water ad libitum. Animals were randomly selected for either the internal carotid injection procedure or the direct inoculation procedure and then were further divided into culture medium only control group, Walker 256 tumour CRCTU group and Walker 256 tumour ATCC group.
DNA fingerprinting of cell lines
The Walker 256 cell lines obtained from the CRCTU and ATCC were submitted to IDEXX RADIL for DNA fingerprinting, using 31 short tandem repeat markers that are rat specific in order to establish the genetic profile of the two cell populations. Cell samples were also tested for cross species contamination.
Internal carotid artery injection
Animals allocated to the internal carotid injection procedure were sacrificed at 24 h (early, n=5), 6 days (intermediate, n=5) and 9 days (late, n=9) for the CRCTU Walker 256 cells, and at 24 h (early, n=5), 4 weeks (intermediate, n=5) and 10 weeks (late, n=9) for the ATCC Walker 256 cells. The selected late time points were determined after a pilot study of tumour burden and animal weight loss for both cell lines. The method for internal carotid artery injection of tumour cells to induce metastatic brain tumour growth has been previously described in detail [
45]. Briefly, under 2% isoflurane inhalation anaesthesia via endotracheal tube, a longitudinal skin incision was made to expose the carotid bifurcation. The ophthalmic artery, superior thyroid artery and pterygopalatine artery were occluded to specifically deliver tumour cells to the brain. The external carotid artery was sacrificed, forming a surgical stump to provide an access point for cannulation of the internal carotid artery for injection of 0.2 mL of culture medium or tumour cell suspension, following temporary occlusion of the common carotid artery. Once the cannula was removed and a suture tied around the external carotid stump, blood flow through the common carotid artery was re-established and the wound sutured.
Direct inoculation
Animals that received direct intraparenchymal inoculation were sacrificed at 7 days and 4 weeks for the CRCTU and ATCC Walker 256 cells, respectively (n=6/group). Direct stereotaxic inoculation of tumour cells into the right striatum for induction of metastatic brain tumour has been previously described in detail [
46]. Briefly, animals were anaesthetised using 3% isoflurane inhalation anaesthesia via a nose cone, placed in a stereotactic frame and a midline scalp incision made to expose the skull. A 0.7 mm burr hole was performed at stereotaxic coordinates: anterior 0.5 mm, lateral 3 mm to the right relative to the bregma. A 30 gauge Hamilton syringe was inserted and lowered stereotaxically 5 mm and 8 μL of culture medium or tumour cell suspension injected directly into the brain tissue over 10 minutes. 5 minutes following inoculation, the needle was removed, the hole was sealed with bone wax and the wound sutured.
Tumour volume
For histological analysis, animals were transcardially perfused with 10% formalin under terminal anaesthesia induced by intraperitoneal administration of pentobarbitone sodium (60 mg/kg). Brains were embedded in paraffin wax and sequential 5 μm coronal sections were cut from blocks 2mm thick in a rostro-caudal direction, to be used for haematoxylin and eosin staining and immunohistochemistry. The haematoxylin and eosin stained slides were scanned using a Nanozoomer (Hamamatsu, Hamamatsu City, Japan) and images used to calculate tumor volume. This was performed by determining the area of tumour in each section using the NDP viewer programme and multiplying the area by the distance between sections as previously described [
47].
Immunostaining
Slides from each model were stained for albumin (ICN Pharmaceuticals, 1:20,000), glial fibrillary acidic protein (GFAP, Dako, 1:40,000) and ionized calcium binding adaptor molecule 1 (IBA1, Dako, 1:50,000). Tumour cells were also grown on cover-slips
in vitro to be immunostained for cytokeratin18 (Gene Tex, 1:3,000). Immunohistochemistry was performed using the standard streptavidin procedure with 3,3
′-diaminobenzidine (DAB) for visualization and haematoxylin counterstaining. Slides were scanned using the Nanozoomer. Albumin immunostaining, expressed as the weighted %DAB in each coronal section, was estimated using colour deconvolution techniques, as described previously [
48,
49]. For GFAP and IBA1 immunoreactivtiy, 4 fields of view were taken from the cortex and striatum for the internal carotid artery injection model and the direct inoculation model. The immunolabelled cells in these images were counted and the mean number calculated for all images from each brain.
Statistical analysis
Results were expressed as mean±SEM and an unpaired t test (for two groups) or a one-way analysis of variance followed by a Bonferroni post test (for more than two groups) performed. Values of p<0.05 were designated as significant.
Competing interests
The authors declare that they have no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations that could inappropriately influence, or be perceived to influence, our work.
Authors’ contributions
We declare that all the authors have approved the submission of this article. We declare that all authors have contributed to scientific work and writing and editing of this article, KML as part of her PhD study, conducted the practical work and writing of the paper; EHW contributed to the discussion and critical reading of the paper; RV co-supervisor of the study, discussion throughout and critical reading and editing of the article; MNG, Principal supervisor of the study, discussion throughout, critical reading and editing the article and final submission.