Establishment and characterization of new mammary adenocarcinoma cell lines derived from double transgenic mice expressing GFP and neu oncogene

A new murine cell line, named GFPneu, was established from a mammary adenocarcinoma arising in double transgenic MMTVneu x CMV-GFP mice. Breast tumours develop in 100% of females after 2 months latency, as a result of the over-expression of the activated rat neu oncogene in the mammary glands. All tissues, and in particular the breast tumours, express the GFP protein. This cell line was tumorigenic when inoculated into nude mice and the derived tumours showed the same histological features as the primaries from which they were isolated. Their histopathology reproduces many characteristics of human breast adenocarcinomas, in particular their ability to metastasize. The GFP marker allows us to visualize the presence of lung metastases in fresh tissues immediately, to confirm the histopathology. From a lung metastatic fluorescent nodule, we derived a further cell line, named MTP-GFP, which we also characterized. These two cell lines could be useful to study the role played by the neu oncogene in the maintenance of the transformed phenotype, in the metastatic process, to test novel therapeutic strategies to inhibit primary tumour growth and to observe the generation of distant metastases.     

Invasion and metastasis.

Malignant tumours cause sickness and death largely because they invade and metastasize. Such spread is made possible by many cellular properties, including the ability of neoplastic cells to move and to release degradative enzymes. These properties enable tumour cells to break free of the primary tumour, penetrate blood or lymphatic vessels and, after being transported to distant sites, pass out of the vessels to establish new tumours. Not all cells in a tumour, however, are able to metastasize, so the process tends to select for greater malignancy in the secondary tumour. The heterogeneity of tumours probably accounts for the difficulty of providing effective treatment, in that the various subpopulations of cells arising from each tumour vary in their responses to chemotherapeutic agents. We do not yet understand the process sufficiently to treat cancer patients by interfering selectively with the metastatic mechanisms.     

The role of PET-CT in detecting unknown primary tumour in patients with cervical lymph node metastases

PET-CT examination was conducted with 440 patients treated at the Department of Head and Neck Surgery, National Institute of Oncology, Budapest, between January 1, 2006 and December 31, 2010. Out of them 77 patients were selected with whom no examination of any sort (physical, pan-endoscopy, or any of the conventional imaging techniques) succeeded in identifying the primary tumour. In each case the primary examination (aspiration cytology and histology) verified cervical metastases, most of them being squamous cell carcinoma. The significance of PET-CT was retrospectively evaluated in cases of unknown primary tumour with verified cervical metastases. We tested the sensitivity of PET-CT in detection of the primary malignant tumour, and possible distant metastases or a second primary in order to plan an optimal treatment schedule for the patient. Patients with whom the examinations specified in the treatment protocol (physical examination, pan-endoscopy, conventional imaging, biopsy) had failed to diagnose the primary tumour were referred to PET-CT. In each case 18F-FDG tracer was used. In 21/77 patients (27%), the PET-CT yielded unequivocal evidence for the primary tumour confirmed by histology, as well. With 10 others (13%), the precarious diagnoses by various imaging techniques were confirmed by the PET-CT. False positive findings with PET-CT that were not verified either by histology or control examination tests occurred but in 10 patients (13%). Concerning the primary tumour, false negative result was obtained only with 3 patients (4%). It should be noted that their retrospective evaluation proved diagnostic errors, the primary tumours were visible in all the scans. With 33 patients (43%) PET-CT furnished no additional information compared to the previous examinations. In 10 patients, asymptomatic distant metastases and in 3 patients synchronous tumours were diagnosed. We also acknowledge that the significance of PET-CT using 18F-FDG is unquestionable in the detection of unknown primary tumours. It is strongly recommended to re-include the detection of unknown primaries in the approved national indication list of PET-CT. (Note, until January 1, 2008 it had been included!) PET-CT is capable of detecting a primary tumour, after all unsuccessful diagnostic examinations till then, in 25-40% of the cases. One cannot disregard the role and significance of PET-CT in the detection of asymptomatic synchronous tumours, or distant metastases. These benefits make PET-CT a suitable tool for the refinement of individually tailored treatment strategies leading to better therapeutic results and more favourable cost-benefit ratio.     

131I]metaiodobenzylguanidine therapy in 20 patients with malignant pheochromocytoma.

Twenty patients, 16 male and 4 female (aged 11-76 years), with metastatic pheochromocytoma were treated in our Institution between 1985 and 1990. Metastases occurred in all patients: at presentation in 11 patients, with a 10 to 30 month delay in 7 patients, and 9 and 28 years later respectively in 2 patients. Catecholamines hyperproduction was present in all patients. Metaiodobenzylguanidine (MIBG) uptake was found in 16 patients after a diagnostic dose and only after a therapeutic dose in 1 patient. Surgery was performed on primary tumor (18 patients) and on distant metastases (10 patients). 131I-MIBG therapy was performed in 11 patients, 9 of whom were evaluable. The cumulative activity ranged from 3.7 to 26.3 GBq (100 to 711 mCi) in 1 to 6 courses. We observed symptomatic improvement (5 patients) and partial tumor response in 2 patients, which lasted for 28 and 9 months respectively, terminating with a rapidly progressing disease involving the bone marrow. Stabilisation was observed in 3 patients. Moderate myelosuppression occurred in 4 patients. Fifteen patients died with a median survival of 16 months (range 3-60). Response to therapy was poor and further evaluation of the presently available therapeutic approaches is needed.     

Hard Wired Genotype in Metastatic Breast Cancer

Recently, we showed by gene-expression profiling that the molecular program established in a human primary breast carcinoma is highly preserved in its distant metastases. According to the predominant model of metastasis, the capacity of a primary tumor to metastasize is acquired only rarely and late in tumorigenesis. Our findings challenge this common theory and imply that the metastatic nature of ‘poor prognosis profile’ breast carcinomas is an inherent feature, and not reserved to advantageous subpopulations.     

Origin of metastases: Subspecies of cancers generated by intrinsic karyotypic variations

Conventional mutation theories do not explain (1) why the karyotypes of metastases are related to those of parental cancers but not to those of metastases of other cancers and (2) why cancers metastasize at rates that often far exceed those of conventional mutations. To answer these questions, we advance here the theory that metastases are autonomous subspecies of cancers, rather than mutations. Since cancers are species with intrinsically flexible karyotypes, they can generate new subspecies by spontaneous karyotypic rearrangements. This phylogenetic theory predicts that metastases are karyotypically related to parental cancers but not to others. Testing these predictions on metastases from two pancreatic cancers, we found: (1) Metastases had individual karyotypes and phenotypes. The karyotypes of metastases were related to, but different from, those of parental cancers in 11 out of 37 and 26 out of 49 parental chromosomal units. Chromosomal units are defined as intact chromosomes with cancer-specific copy numbers and marker chromosomes that are > 50% clonal. (2) Metastases from the two different cancers did not share chromosomal units. Testing the view that multi-chromosomal rearrangements occur simultaneously in cancers, as opposed to sequentially, we found spontaneous non-clonal rearrangements with as many new chromosomal units as in authentic metastases. We conclude that metastases are individual autonomous species differing from each other and parental cancers in species-specific karyotypes and phenotypes. They are generated from parental cancers by multiple simultaneous karyotypic rearrangements, much like new species. The species-specific individualities of metastases explain why so many searches for commonalities have been unsuccessful.     

Neuron specific enolase-positive breast carcinomas

Ninety-eight patients treated for breast carcinomas were followed from 54 to 75 months after primary diagnosis. All had undergone a modified radical mastectomy with removal of axillary lymph nodes. 36 breast carcinomas were NSE-positive and 62 were negative. NSE-positive tumours were significantly more frequently estrogen receptor-positive than the NSE-negative tumours, and the estrogen receptor values were higher in the NSE-positive groups. Patients with NSE-positive tumours and patients with NSE-negative tumours did not differ with regard to presence of lymph node metastases at the time of primary surgery. However, the study showed that patients with NSE-positive tumours had a tendency towards more lymph node metastases after primary surgical intervention, but a better outcome than patients with NSE-negative tumours and metastases. This study, with a 5-year follow up, failed to demonstrate any major prognostic significance of immunostaining for NSE.     

The immunohistochemical expression of stress-response protein (srp) 60 in human brain tumours: relationship of srp 60 to the other five srps, proliferating cell nuclear antigen and p53 protein

This study analyzed the expression of stress-response (heat-shock) protein 60 (srp 60) in a series of 158 human brain tumours. Immunohistochemical procedures were employed; cells of the human cervical cancer line HeLa S3 exposed to hyperosmolar stress served as positive controls. Deposits of reaction products were found in the cytoplasm. Approximately half of the glioblastomas multiforme (17/31), breast carcinoma metastases (6/10), and lung carcinoma metastases (5/11) as well as about one-third of the astrocytomas (5/13) and meningiomas (8/23) had tumour cells that expressed srp 60. A positive reaction for srp 60 was also seen in some medulloblastomas (2/16), primitive neuroectodermal tumours (PNETs) (2/11), schwannomas (2/21), and pituitary adenomas (2/7), but no positive reactions were observed with oligodendrogliomas and ependymomas. Compared with srp 60-negative tumours, srp 60-positive tumours coexpressed one or more stress-related proteins, among which srp 90, srp 72, srp 27, alphaB-crystallin and ubiquitin occurred with higher frequencies; a high correlation between srp 60 and the other five srps (0.88 - 0.97, p<0.01, Pearson correlation coefficient) was observed in srp 60-positive tumours. In contrast, the correlation coefficient in srp 60-negative tumours was not significant (-0.26 - 0.71). There was a tendency for the proliferating cell nuclear antigen (PCNA)-labeling index to be higher in glioblastomas, astrocytomas, medulloblastomas, PNETs, and breast and lung carcinoma metastases that expressed srp 60 than in those that did not. No significant immunohistochemical reactions of srp 60, PCNA and p53 protein were seen with sections of normal brain tissues. We conclude that primary and metastatic tumours of the brain produce srp 60 and that srp 60 in certain brain tumour cells may coexpress the other five srps. In addition, srp 60 expression might depend, in part, on proliferating potential.     

Feasibility and limits of an orthotopic human colon cancer model in nude mice

We sought to develop an accurate colorectal cancer model in nude mice with stable local growth, tumor cell dissemination, and reproducible metastatic capacity. To this end, we orthotopically transplanted histologically intact human colorectal cancer tissue from 10 human patients into nude mice. After successful local tumor growth, tumor tissues were retransplanted as many as 9 times in serial passage. All specimens were transplanted using microsurgical techniques. Histologic, immunohistochemical, and polymerase chain reaction techniques were used to determine tumor growth rates and kinetics, development of regional lymph node and distant hepatic metastases, and the induction of minimal residual disease (MRD). Stable local tumor growth rates with variable growth kinetics were detected in 73.4% of all mice. The lymph node and hepatic metastasis rates were low, at 18.4% and 4.9%, respectively. MRD, as reflected by CK20 positivity of the bone marrow in animals with lymph node and hepatic metastases, was present in 22.2%. The orthotopic colorectal cancer model described here is feasible for the induction of reproducible local tumor growth but is limited by variable growth kinetics and the low rate of lymph node and hepatic metastases. Cytokeratin-positive cells indicative of MRD could be detected in the bone marrow of approximately 25% of the nude mice with metastases. The observed induction of MRD after orthotopic implantation of intact human colon cancer in animals with lymph node and hepatic metastases might be improved if established colon cancer cell lines were used.     

Organ-Specific Metastases in Melanoma: Experimental Animal Models

Metastases from certain primary tumors frequently exhibit specific organ preference. Animal models have been developed to induce in a reproducible fashion the formation of organ-specific metastases by malignant melanoma cells. Some of these models rely on the use of immunodeficient mice, which can support the growth of murine as well as human malignant melanomas. Moreover, immunodeficient mice, because of their diminished ability to mount an effective immune response, allow the expression of malignant properties (e.g., preferential colonization of certain organs), which are intrinsic to transplanted melanoma cells. This review discusses the relevant factors (and limitations) of some of the animal models used to study the in vivo properties of melanoma cells.     

Murine mammary tumor cells with a claudin-low genotype

In adults, bone is the preferential target site for metastases from primary cancers of prostate, breast, lungs and thyroid. The tendency of these cancers to metastasize to bone is determined by the anatomical distribution of the blood vessels, by the genetic profile of the cancer cells and by the biological characteristics of the bone microenvironment that favour the growth of metastatic cells of certain cancers. Metastases to bone may have either an osteolytic or an ostoblastic phenotype. The interaction in the bone microenvironment between biological factors secreted by metastatic cells, and by osteoblasts and osteoclasts, and the osteolytic and osteoblastic factors released from the organic matrix mediate a vicious cycle characterized by metastatic growth and by ongoing progressive bone destruction. This interaction determines the phenotype of the metastatic bone disease.     

A novel model for the investigation of orthotopically growing primary and secondary bone tumours using intravital microscopy

Here is reported the development of an experimental model using intravital microscopy as a tool to orthotopically investigate malignant bone tumours. Although up to 85% of the most frequently occurring malignant solid tumours, such as lung and prostate carcinomas, metastasize into the bone, and despite the knowledge that a tumour's course may be altered by its surrounding tissue, there is no adequate experimental model available enabling the investigation of orthotopically grown bone tumours in vivo. Intravital microscopy is an internationally accepted experimental method, used in various acute and chronic animal models, that enables qualitative and quantitative analysis of the angiogenesis, microcirculation, growth behaviour, etc. of various benign and malignant tissues. Non-invasive investigations of up to several weeks are possible. Additionally, tissue samples can be taken after termination of the in vivo experiments for further ex vivo investigation (histology, immunohistochemistry, molecular biology, etc.), elucidating the mechanisms that underlie the in vivo observations. Severe combined immunodeficient mice were fitted with a cranial window preparation where the calvaria served as the site for orthotopic implantation of the solid human tumours Saos-2 osteosarcoma (primary) and A 549 lung carcinoma and PC-3 prostate carcinoma (secondary). In all preparations, the take rate was 100%. Histological assessment confirmed the data obtained in vivo, showing typical tumour growth with infiltration of the surrounding osseous and soft tissues. This novel model serves as a valuable tool in understanding the biology of primary and secondary bone tumours in physiological and pathophysiological situations, with implications for the most areas of tumour therapy such as chemotherapy, radiation and antiangiogenesis.     

Quantitative comparison of three C. parvum strains for immunotherapy of transplanted rat tumours

Summary Preparations of three commercially available killed bacterial suspensions designated Corynebacterium parvum have been compared for immunotherapy of transplanted rat tumours. The Wellcome CN6134 preparation was quantitatively superior to Institut Merieux IM1585 in suppressing local, subcutaneous, growth of tumour when injected in admixture with cells, for the control of thoracic malignant effusions when administered intrapleurally and for active specific immune stimulation where incorporated into vaccines of irradiated cells. The Wellcome CN5888 preparation was virtually ineffective. These observations, together with the recent re-classification of C. parvum into three distinct species of the genus Propionibacterium, i.e., P. acnes (Wellcome CN6134), P. granulosum (Wellcome CN5888), and P. avidum (Institut Merieux IMI1585) emphasize that the simple designation C. parvum is inadequate for the vaccines currently available for experimental or, more importantly, clinical immunotherapy.     

Das Rätsel der Metastasierung

The riddle of the metastatic process Despite all progress in the understanding of the molecular basis of malignant tumours, the chances for being cured has not changed dramatically for the better in the last decades for many clinically important cancers as those of lung, pancreas, colon and ovary. The lack of success can be attributed to the early formation of distant metastases, which can generally not be cured. Without a deeper understanding of the metastatic process, it will be difficult to achieve therapeutic success. An additional problem is posed by the fact, that larger primary tumours as well as metastases develop an interstitial fluid pressure, which hinder the influx of drugs into them. This problem creates an additional obstacle for successful cancer therapy.     

New variants of B16 mouse melanoma: differentiation and metastatic properties

A system of tumor transplantation has been developed to select metastatic variants of B16 in mutants of the C57BL/6J black strain of mice. The effects of transplantation into nonagouti a/a and mutant recipients on the production of melanin and on the metastatic potential of tumors were investigated. Transplantation of the pigmented B16 melanoma from a nonagouti black a/a host to a yellow mutant Ay/a recipient resulted in an achromic and metastatic variant melanoma, designated YB16. The amelanotic phenotype occurred consistently after more than ten passages through yellow mice and simultaneously with an increase in the incidence of pulmonary metastases. When YB16 was transplanted back to the nonagouti black a/a host, a second variant, MB16, characterized by its variable pigmentation, was obtained. Pigmented and/or entirely achromic tumors were observed. MB16 was dramatically more metastatic than B16 and YB16 when injected s.c. or i.v. Metastases in the lungs were pigmented and/or achromic. The properties of tumor cells derived from artificially induced metastases were investigated after s.c. and i.v. injections. Whereas the metastatic cells expressed a potent ability to generate metastases when injected s.c., no differences in the incidence of metastases, as compared to the metastatic potential of cells of parental origin, were observed after i.v. injection. In the MB16 variant, there appeared to be an inverse relationship between differentiation (production of melanins) and malignancy. Our results demonstrate that differentiation and metastatic behaviour are dependent on specific mutations in the host environment which generate a pool of tumor cells from which highly metastatic variants can be selected.     

Roles of p38 MAPKs in invasion and metastasis

Cells from primary tumours need to go through several steps to become fully metastatic. During this process, cancer cells acquire the ability to invade, migrate across the surrounding tissue, enter into the circulation and colonize distant organs. In the present paper, we review recent progress in understanding how the p38 MAPK (mitogen-activated protein kinase) signalling pathway participates in the different steps of metastasis. Experimental evidence suggests that tumour cells need to modulate p38 MAPK activity levels to successfully metastasize.     

Growth of human pheochromocytomas in the anterior eye chamber of the rat. A histochemical study on amine and peptide content of pheochromocytoma tumour cells

Tissue pieces from seven benign human pheochromocytomas have been successfully transplanted to the anterior eye chamber of cyclosporin-treated rats. In vivo observations showed that 74-99% of the tumour transplants were vascularized within one to two days after transplantation. No increase in the size of the transplants was noted during the observation period (1-4 weeks). Tumour transplants grown in non-immunosuppressed rats were initially vascularized but rejection started to occur one week after transplantation. Histochemical analysis of tumour transplants grown in immunosuppressed rats demonstrated numerous tumour cells with strong catecholamine fluorescence, some of which formed long cell processes on the host iris. Immunocytochemical analysis of tumour transplants demonstrated positively labelled tumour cells after incubation with antisera against neuropeptide Y, enkephalin, vasoactive intestinal polypeptide, somatostatin, substance P, dopamine-beta-hydroxylase, tyrosine hydroxylase and serotonin. A similar histochemical and immunocytochemical pattern was observed in primary tumours but tumour cells sending out cell processes were observed less frequently. Human pheochromocytomas may thus be successfully grown in oculo in cyclosporin-treated rats. This may prove to be a suitable model for the study of storage and release of catecholamines and neuropeptides from pheochromocytoma tumour cells.     

Local and metastatic growth of Lewis lung carcinoma as a function of its transplantation site. Application to the study of the pharmacology of sulphadiazine triazene]

The presence of a local tumour and pulmonary metastases is studied after the administration of the Lewis Lung Carcinoma at different sites of transplantation. The intravenous administration routes, in the tail vein and in the portal vein, injections in the liver and in the muscle of the left hindleg are used. The injection in the tail vein induces pulmonary "metastases". The injection in the portal vein is followed by multiple tumours in the whole liver and pulmonary metastases. An unilobar hepatic tumour and early pulmonary metastases appear after transplantation in the liver. Intra-muscular injection gives a local tumour which can be weighed after amputation of the leg and pulmonary metastases. A test of treatment by Sulphadiazine Triazene points out a weak action on the primary tumour and a larger one on the metastases.     

Haematuria as a presentation of metastatic oesophageal carcinoma

Haematuria is a classical symptom of urological disease often signifying a primary bladder cancer. Rarely, however, the presence of blood in the urine can be due to secondary spread of tumours into the bladder from distant sites. Notably this has been reported to occur in breast cancer, malignant melanoma and gastric cancers. Haematuria due to spread from a primary oesophageal cancer to the bladder has never been reported. We present a case of haematuria confirmed histologically to be due to metastases from a primary oesophageal tumour. Oesophageal cancer is capable of spread to all three neighbouring compartments (abdomen, chest and neck) and therefore has the potential to spread to unusual sites. Clinicians should always carefully regard haematuria in a patient previously treated for cancer and retain a high index of suspicion for distant metastases as being the cause.     

Computational Modelling of Metastasis Development in Renal Cell Carcinoma

The biology of the metastatic colonization process remains a poorly understood phenomenon. To improve our knowledge of its dynamics, we conducted a modelling study based on multi-modal data from an orthotopic murine experimental system of metastatic renal cell carcinoma. The standard theory of metastatic colonization usually assumes that secondary tumours, once established at a distant site, grow independently from each other and from the primary tumour. Using a mathematical model that translates this assumption into equations, we challenged this theory against our data that included: 1) dynamics of primary tumour cells in the kidney and metastatic cells in the lungs, retrieved by green fluorescent protein tracking, and 2) magnetic resonance images (MRI) informing on the number and size of macroscopic lesions. Critically, when calibrated on the growth of the primary tumour and total metastatic burden, the predicted theoretical size distributions were not in agreement with the MRI observations. Moreover, tumour expansion only based on proliferation was not able to explain the volume increase of the metastatic lesions. These findings strongly suggested rejection of the standard theory, demonstrating that the time development of the size distribution of metastases could not be explained by independent growth of metastatic foci. This led us to investigate the effect of spatial interactions between merging metastatic tumours on the dynamics of the global metastatic burden. We derived a mathematical model of spatial tumour growth, confronted it with experimental data of single metastatic tumour growth, and used it to provide insights on the dynamics of multiple tumours growing in close vicinity. Together, our results have implications for theories of the metastatic process and suggest that global dynamics of metastasis development is dependent on spatial interactions between metastatic lesions.