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
Human Cell Lines |
Breast cancer |
MDA-MB 231, MCF-7, ZR75 |
Colon |
HT29, HCT 116, WiDR, HCA7 |
Head and neck |
PE/CA-PJ41, PE/CA-PJ15, FaDu, Cal29 |
Leukemia |
K562, THP-1 (monocytic leukemia), HL60 (myelod leukemia), Jurkat (leukemic T cell), MV4-11 (acute monocytic) |
Liver |
HepG2, Lx2 (hepatic stellate cell line), HuH7 |
Lung |
A549, H1975, H1650, H358, LCLC-LO3H, HCC-15, LOU-NH91 |
Melanoma |
A2058 (HT168, HT168-M1), HT199, MeWo, WM983A, WM983B, WM35, M24met |
Ovarian |
OVCAR-8, OVCAR-3, SK-OV-3, A2780 |
Prostate |
PC3, LNCaP (androgen +), DU145 |
Other human cell lines |
HUVEC (umbilical vein endothelial), ECV 304 („immortalised” HUVEC), KS IMM (Kaposi sarcoma endothel), HT1080 (fibrosarcoma), HeLa (cervix), KB („keratin-forming” HeLa), U87 (glioblastoma), U87-MG vIII (EGFRvIII transfectant), A431 (epidermoid carcinoma), Panc-1 (pancreas), MRC-5 (fibroblast from lung) |
Mouse Cell Lines |
Mouse cell lines |
B16a (melanoma, amelanotic), B16 (F0, F10), LLT-HH (Lewis lung, high liver preference in metastasis), MXT (breast cc), CMT 93 (rectum cc), LLT (Lewis lung), C38 (colon), C26 (colon), S180 (sarcoma), P388 (leukemia) , L929 (fibroblast), 4T1 (breast) |
Metastasis model systems and orthotopic transplantations (samples)
I. Brain metastases
1. Macroscopic meatastases are produced by injecting tumor cells directly in the brain parenchima
Tumor lines |
host |
duration (days) |
C38 mouse colon carcinoma |
C57Bl/6 |
7-10 |
HT1080 human fibrosarcoma |
SCID |
7-10 |
ZR75 human mammary carcinoma |
SCID |
28 |
HT25 human colon carcinoma |
SCID |
28 |
H1650 human NLSC |
SCID |
28 |
2. Microscopic metastases are produced by injecting tumor cell in the carotid artery
Tumor lines |
host |
duration (days) |
3LL-HH mouse lung carcinoma |
C57Bl/6 |
10-14 |
B16 mouse melanoma |
C57Bl/6 |
10-14 |
A2058 human melanoma |
SCID |
28 |
II: Pleural metastases can be produced by injecting tumor cell in the thoracic cavity. Metastases can be analyzed by dissecting the diaphragm
Tumor lines |
host |
duration (days) |
3LL-HH mouse lung carcinoma |
C57Bl/6 |
7-10 |
SPC111 human mesothelioma |
SCID |
28-35 |
III. Colon carcinoma: Tumor pieces are transplanted onto the serosal surface of the caecum
Tumor lines |
host |
duration (days) |
metastasis |
C-26 mouse colon carcinoma |
BALB/c |
14-18 |
liver, LN |
HT29 human colon cc. |
SCID/NSG |
45-50 |
liver, lung, LN |
IV. Mammary carcinoma (injected into mammary fat pad)
Tumor lines |
host |
duration (days) |
metastasis |
MXT mouse mammary cc. |
BDF1 |
25-30 |
lung, axillary LN |
MDA-MB-231 human |
SCID/NSG |
80-90 |
lung, axillary LN |
V. Prostate carcinoma (tumor cells are injected into the prostate)
Tumor lines |
host |
duration (days) |
metastasis |
PC3 human prostate cc. |
SCID, NSG |
25-30 |
|
LNCAP human prostate cc. |
SCID, NSG |
25-35 |
|
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.
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