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 .
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 . 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. 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 . 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 . 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.
Walker 256 carcinoma is rat mammary tumour cell line that originally occurred spontaneously in a pregnant albino Sprague-Dawley rat . 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 , 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 . 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.