Naturally fluorescent proteins have revolutionized biology by enabling what was formerly invisible to be seen clearly. These proteins have allowed us to visualize, in real time, important aspects of cancer in living animals, including tumour cell mobility, invasion, metastasis and angiogenesis. These multicoloured proteins have allowed the colour-coding of cancer cells growing in vivo and enabled the distinction of host from tumour with single-cell resolution. Visualization of many aspects of cancer initiation and progression in vivo should be possible with fluorescent proteins.

http://www.metamouse.com/RMH_2005.Nature_Reviews_Cancer.pdf

The multiple uses of fluorescent proteins to visualize cancer in vivo

The revolution of in vivo cancer biology enabled by fluorescent proteins is described. The high extinction coefficients, quantum yields, and unique spectral properties of fluorescent proteins have been taken advantage of in order to visualize, in real time, the important aspects of cancer in living animals, including tumor cell trafficking, invasion, metastasis, and angiogenesis. Fluorescent proteins enable whole-body imaging of tumors on internal organs. These multicolored proteins have allowed the color-coding of cancer cells growing in vivo with distinction of different cell types, including host from tumor, with single-cell resolution.

https://www.spiedigitallibrary.org/journals/Journal-of-Biomedical-Optics/volume-10/issue-04/041202/Advantages-of-multi-color-fluorescent-proteins-for-whole-body-and/10.1117/1.1992485.pdf

Advantages of multi-color fluorescent proteins for whole-body and in vivo cellular imaging

The level of TNFalpha expression is increased after partial hepatectomy, and experimental evidence exists that TNFalpha plays a key role in liver regeneration. Contradictory results are reported about the influence of TNFalpha on tumor growth: on the one hand, stimulation of tumor growth in various animal models and, on the other hand, intraperitoneally administered TNFalpha leads to reduced metastasis formation. TNFalpha may be one responsible factor for increased metastasis formation after surgical trauma. The objective of our study was to clarify the influence of TNFalpha on the formation of liver metastases in a syngenic mouse model in vivo. We used a novel marker system, EGFP transfected C26 tumor cells for in vivo observation of metastasis formation by intravital microscopy. We analyzed the effect of intraperitoneal TNFalpha-injection on tumor cell adhesion, extravasation and tumor development. The expression of ICAM-1, VCAM-1 and E-Selectin was measured by Western blot and immunohistochemical staining. Tumor load was assessed by determining EGFP in Western blots. GdCl(3) was employed 24 and 48 hr before tumor cell injection to selectively deplete the liver of functioning Kupffer cells. We observed significantly more extravasated tumor cells in the TNFalpha-pre-treated animals at early time points with increased expression of adhesion molecules. Measurement of the EGFP levels showed fewer liver metastases in the TNFalpha-pretreated animals at day 8. After GdCl(3) pretreatment even lower levels of EGFP, i.e., fewer metastases and also lower expression levels of ICAM-1, VCAM-1 and E-Selectin could be observed. TNFalpha, acts in a bidirectional manner: whereas TNFalpha facilitates tumor cell adhesion and extravasation of C26 tumor cells by inducing the expression of adhesion molecules, at later time points, TNFalpha seems to hinder the formation of liver metastases.

Influence of TNFA on the formation of liver metastases in a syngenic mouse model.

Metastases are the leading cause of treatment failure and death of patients affected by malignant tumors, which makes them a major therapeutic target. During the last decade, efforts made to understand the mechanisms governing the passage of a localized tumor cell to a metastatic one has led to significant advances in this field. In vivo models using nude mice largely contributed to the understanding of this multi-step phenomenon, thus allowing the discovery of new targets and the development of therapeutic agents. These models were however hampered by the difficulties to detect micrometastases. The recent introduction of the green fluorescent protein (GFP) as a marker for tumor cells has radically changed the use of these models, in particular by allowing detection of single cell metastases in vivo. In this review, we discuss the major advantages of models using GFP-labeled cells and their limits.

/pdf/metastasis-models-the-green-fluorescent-revolution-2h7q4p3req.pdf

Metastasis models: the green fluorescent revolution?

We investigated the therapeutic efficiency of sulfonate-modified polyvinyl alcohol beads loaded with doxorubicin, irinotecan or mitoxantrone in vitro and in vivo in a model of experimental peritoneal carcinomatosis (PC). In vitro, cell proliferation was efficiently impaired by doxorubicin drug eluting bead (DEB) treatment while mitoxantrone DEBs were less effective than. Irinotecan showed little effect for both DEBs and free drug. Apoptosis was not different between free mitoxantrone and the DEB form while more apoptosis induction was observed in cells incubated with free doxorubicin and irinotecan. Experimental PC was produced in mice. The therapeutic efficiency of either mitoxantrone and doxorubicin DEB or free drugs were compared. Mice were treated either once on day 12 or by 3 repetitive applications on days 7, 10 and 12. Mice treated by DEBs showed less weight loss and mortality. Therapeutic effect was determined by measuring tumor volume and tumor load on the day 15 after tumor inoculation. For the single application on the day 12, an advantage could be observed for the free drugs. After 3 repeated injections of both free and mitoxantrone DEB no difference in tumor load or tumor volume could be observed. Least tumor load and tumor volume was observed in mice that received 3 repeated injections of doxorubicin DEB. No animal survived 3 injections of free doxorubicin. We conclude that bead encapsulation of chemotherapeutic drugs may show the advantage of less toxicity in peritoneal spread of colorectal cancer.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/ijc.24211

Doxorubicin and mitoxantrone drug eluting beads for the treatment of experimental peritoneal carcinomatosis in colorectal cancer

Many animal models for metastatic colorectal cancer represent clinical manifestations just inaccurately. We introduce a novel mouse model for metastastatic colorectal cancer. In order to remain close to the clinical disease a syngenic murine colon carcinoma cell line (colon 26 cells) was transfected with enhanced green fluorescent protein (EGFP). The transfected cells maintain the highly malignant attributes of the wild-type cells. Following injection into the portal circulation of Balb/c-mice, liver metastases occur in the same time span. Using the fluorescent attributes of the transfected cells, an approximation of the tumor load in liver tissue can be achieved by fluorescence activated cell sorting (FACS) and fluoroscan analysis. Tumor cell load in liver tissue can be accurately measured by Northern blot and Western blot analysis of liver tissue containing EGFP-transfected colon cancer metastases (1250 cells/mg liver tissue and 1000 cells/mg liver tissue) respectively. Confocal microscopy and intravital microscopy confirmed the growth of tumor metastases, originating from the intravascular compartments. The presented animal model using EGFP-transfected colon 26 cells allows the detecting of tumor growth in vivo and post mortem, as well as an accurate quantification of the tumor load in the liver tissue.

Hongyue Zhang

Papers

Enhanced green fluorescent protein-transfection of murine colon carcinoma cells: key for early tumor detection and quantification.