In-vivo models of metastasis

Spontaneous models of metastasis.
(tumor cells have to complete all steps of the metastatic process)
Site of injection (target organs): subcutaneous tissue (lung), spleen (liver), hind leg muscle (lung). 
Orthotopic transplantation of tumor cells (target organs): wall of large bowel (liver), kidney (lung), mammary fat pad (lung), dermis (lung and/or lymphnodes).

Experimental models of metastasis:  
Site of injection: tail vein, portal system, carotid artery, left ventricle of the heart. Target organs are: lung, liver, brain, all organs, respectively.

In-vitro models of the steps of the metastatic process.

Tumor cell lines used: human and mouse melanomas, fibrosarcomas, colon carcinomas, lung carcinomas, breast carcinomas, prostate carcinomas, lymphomas, HNSCC.

Tissue invasion

2D models:

• confluent cell cultures of normal cells (fibroblasts, hepatocytes, different epithelial cells) confronted with tumor cell of different origin.
• adhesion of tumor cells to cryosections from different organs

3D models:

• organ cultures/regenerated organ fragments (chorioallantoic membrane, diaphragm, human amnion, embryonic skin, lung cubes.

Intravasation. isolated brain capillaries are confronted with different tumor cell lines. 
Extravasation. interaction of cultures of endothelial cells or isolated vessel fragments with tumor cells.

Analyzation of tumor cell motility

Types of cellular motility: chemotaxis (liquid phase gradient), haptotaxis (solid phase gradient), chemokinesis (random motility).

Migration tests.

2D: cell culture wounding, phagokinetic track assay, videomicroscopy, emigration of cells from tumor spheroids (scatter).
3D: Boyden chamber (cell number is determined on the lower side of the 8- μm pore size membrane), migration into extracellular matrix gels (collagen I, fibrin, basement membrane), organ cultures (chorio-allantoic membrane, denuded cornea).

Assessment of anti-angiogenic potential.

Frozen tumor samples growing at different location are cryosectioned. Immunoflorescence staining is performed to highlight basement membrane and CD31 to unequivocally detect vessels. Vessel density (blood, lymphatic) vessel size, area fraction are determined using image analysis systems.

Assessment of anti-metastatic potential.

Metastases of different tumor lines are produced in spleen-liver, muscle-lung, skin-lung models. The removed target organs are fixed and number and size of the surface colonies are determined. If necessary the target organs are serially sectioned and the area fraction of the metastatic colonies is determined.

Patient derived tumor xenografts (PDTX)

Patient derived tumor xenografts (PDTX) are created when cancerous tissue from a patient’s primary tumor is implanted directly into an immunodeficient mouse. Tumor models including xenografts in mice were used previously mostly in the development of new anticancer drugs. Nowadays xenografts from direct patient-derived tumor tissues (PDTT) in immunodeficient mice yield better models than experimental tumors originating from cell cultures. The new method enables researchers to observe heterogeneous tumor cells with their surrounding tissue elements and matrices representing the clinical situation in humans much better. The cells in PDTX tumors are alive and functionally active through several generations after serial transplantation. Therefore using these models we may investigate tumor response to different therapies, the selection of resistant cell populations and the formation of metastasis predicting the outcomes in the personalized therapy.

Sample Processing and evaluation

● Pathological sample processing

● Microscopic (light, confocal and electron) analysis of samples (FFPE, frozen sections)

● molecular biological techniques

Cell Line

Metastasis model systems and orthotopic transplantations (samples)

I. Brain metastases

1. Macroscopic meatastases are produced by injecting tumor cells directly in the brain parenchima

2. Microscopic metastases are produced by injecting tumor cell in the carotid artery

II:  Pleural metastases can be produced by injecting tumor cell in the thoracic cavity. Metastases can be analyzed by dissecting the diaphragm

III. Colon carcinoma: Tumor pieces are transplanted onto the serosal surface of the caecum

IV. Mammary carcinoma (injected into mammary fat pad)

V. Prostate carcinoma (tumor cells are injected into the prostate)

VI. General metastasis model:
Tumor cells are injected into the left ventricle of the heart. Metastases are formed in all organs

.
VII. Colonization models:
Tumor cells are injected into the tail vein, or spleen, and tumor colonies are formed in lung and liver, respectively.

VIIa. Amputation metastasis model which is used to accelerate metastasis formation:
Tumor cells are injected into the foot pad. The injected leg is amputated after primary tumor is formed. Metastases develop in the lungs.