Abstract
Studies over the past decade have clearly shown that s.c. implant of primary and cultured tumor cells rarely leads to the occurrence of metastatic disease. Orthotopic transplantation of cell suspensions, surgical orthotopic implantation (SOI) of cancer tissue fragments resulted in metastases in many cancer types reaching 100% successful rate. We compared two metastatic models – heterotopic model of Lewis lung cancer and orthotopic B16 mouse melanoma. Both models were syngeneic with high metastatic ratio in C57BL/6 mice after transplantation of cancer cells, by injection into subcutaneous region of mice tail and without surgical intervention. The conclusion is that the localisation of cancer cell injection is a crucial condition for metastatic potential. The site with 100% haematogenous and lymph metastasis rate, after simple injection of cancer cells only, has been defined in mice, without dependence on the genetically predisposition and tumor cell line.
Metastasis is the major cause of cancer deaths and there is a need for clinically relevant in vivo models. For decades, xenografts using well-established human tumor cell lines have been the most commonly used models to study human cancers in mice. Historically, transplantable tumor models were characterized by and selected for rapid primary tumor growth at subcutaneous (s.c. -heterotopic) sites. In this setting, it was uncommon to observe spontaneous metastasis to distant sites (1).
Orthotopic transplantation refers to the delivery of cancer cells to the anatomic location or tissue from which a tumor was derived. Experimental evidence suggests that orthotopically transplanted tumours may be more appropriate models to investigate these physiological strategies than the usual s.c. transplanted tumour models (2, 3).
Here we compared two metastatic models – heterotopic model of Lewis lung cancer and orthotopic B16 mouse melanoma. We observed metastases in C57BL/6 mice after transplantation of cancer cells by injection and without surgical intervention.
Materials and Methods
Cell culture. Cell lines used in this study have been described previously (4). Lewis lung cancer cell line was labeled by GFP (green fluorescent protein) and B16 melanoma with GFP and RFP (red fluorescent protein). Except where noted, cell lines were grown in RPMI 1640 supplemented with 10% fetal bovine serum and gentamicin to 70–80% confluence as described previously (4).
Subcutaneous tumor growth. All of the animals were maintained in a barrier facility. All animal experiments were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals under assurance of Directive 86/609/EEC on the protection of animals used for scientific purposes in the Czech Republic. Three C57BL/6 mice, 6 weeks of age, were injected s.c. with single dose of 2×106 Lewis lung cancer cells and three C57BL/6 mice, 6 weeks of age, were injected s.c. with single dose of 2×106 B16 mouse melanoma. Cells were first harvested by trypsinisation and washed three times with cold serum-free medium and then injected - total volume of 0.2 ml.
Tumor transplantation of C57BL/6 mice. Tumors derived from the Lewis lung cancer and B16 melanoma s.c. tumor growing in the C57BL/6 mice were homogenized and a single cell suspension was prepared. Cells adjusted to appropriate concentration were injected to the subcutaneous space of tail approx. 1 cm from root of the tail. Cells were transplanted in 5×106/mL cells in volume 0,1mL into dorsal side of tail. Animals were anesthetized by ketamine and xylazine during transplantation.
Analysis of metastasis. Mice were divided into two groups according to the tumor type. Animals from both groups were sacrificed four weeks after the Lewis lung cancer and B 16 melanoma injection. The size of primary heterotopic tumor was measured. The tumor volume was counted – Tw=a(b2)/2 (where a and b are tumor length and width in (mm), respectively (5). The tissue samples from lymph nodes and organs, were collected and the presence of micro- and macrometastases was assessed. At least three micrometastatic and one macrometastatic lession per organ or lymph node was needed to be present for an organ to be considered positive for metastasis. The total metastases number was correlated to the tumor volume. The origin of a single fluorescent tumor cell was tested in inverted fluorescence microscope. Whole-body images were obtained by placing the mice in a fluorescent light box equipped with a fiberoptic light source of 490 nm (Lighttools Research, Encinitas, CA) and imaged using a Nikon Coolpix 5000 camera.
Results
A high metastatic rate was determined according to the estimated tumor volume in both observed mouse groups. Metastatic process was declared for each mouse in the groups and the mice were sacrificed four weeks after s.c. tail injection. The comparison of heterotopic and orthotopic metastasis was assessed – quantity, localization and size of the metastases. The average size of primary tumor was comparable – 167 mm3 in heterotopic Lewis lung cancer and 173 mm3 for orthotopic B16 mouse melanoma. The state of detected micro- and macrometastasis is shown in Table I.
Discussion
Studies over the past decade have clearly shown that s.c. implant of primary and cultured tumor cells rarely leads to the occurrence of metastatic disease. Injecting tumor cell suspensions into the analogy on or orthotopic mouse sites occasionally allowed relevant metastases.
Orthotopic transplantation of cancer cells may come from direct injection of tumor cells or the surgical implantation of intact fragments of tumor. Orthotopic injection of cell suspensions is an improvement over simple subcutaneous implantation but the technique has several major disadvantages. On the other side for many orthotopic models the use of surgical orthotopic implantation (SOI) of fragments improves the reproducibility and metastatic outcome within the model (6, 7).
In a comparison of SOI with orthotopic transplantation of cell suspensions, SOI of cancer tissue fragments resulted in metastases in many cancer types in 100% of the nude mice with extensive primary tumor growth (8). The primary tumors resulting from SOI were larger and much more locally invasive than primary tumors resulting from orthotopic transplantation of cell suspension. SOI generated higher metastatic rates than orthotopic transplantation of cell suspensions. Median survival time in the SOI model was significantly shorter than that of orthotopic transplantation of cell suspensions. Histological observation of the primary tumors from the SOI model demonstrated a much more rich vascular network than the orthotopic transplantation of cell suspension. Lymph node and lung metastases were larger and more frequent in the SOI model compared to the orthotopic transplantation of cell suspension models.
In our study, we observed a high metastatic rate of lymph and visceral metastasis after injecting of the tumor cell suspension in C57BL/6 mice in both tumor syngeneic versions – between the orthotopic and heterotopic tumors no significant differences in metastatic ratio, in metastases localisation and numbers have been observed. Without any surgical intervention a high metastatic rate of lymph and visceral metastases has been declared only after injection of tumor cells.
We declare that the most important for the metastatic potential are local conditions which can probably be more important that histological specification of primary tumor. Transplantation of cancer cells into region with limited growth and expansion possibility leads to the early metastatic process into lymphatics and visceral organs. If the space for the primary tumor growth is limited, the tumor proliferation potential prevail above the genetically predisposition of tumor to grow only in predisposed sites. The lymph and hematogenous metastatic ratio is 100% after inject transplantation for both cancer cell lines. Similar aspects were observed in our earlier studies where the primary tumor has been localized between skin and cartilage of mouse ear (9, 10). The metastatic ratio B16 and Lewis lung cancer has been similar in these studies.
The conclusion is a that the localisation of primary tumor in mouse metastatic model is crucial condition for metastasis development. There are sites on mouse body, where after injection of cancer cells 100%-rate of haematogenous and lymph metastasis occurs thanks to the limited space for primary tumor growth.
Acknowledgements
Grant KONTAKT ME 10045, Ministry of Education, Youth and Sports and Grant IGA NS 9976/3, Ministry of Health Czech Republic.
- Received July 4, 2011.
- Revision received August 19, 2011.
- Accepted August 22, 2011.
- Copyright © 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved