Metastasis and Circulating Tumor Cells

Abstract

Cancer is a prominent cause of death worldwide. In most cases, it is not the primary tumor which causes death, but the metastases. Metastatic tumors are spread over the entire human body and are more difficult to remove or treat than the primary tumor. In a patient with metastatic disease, circulating tumor cells (CTCs) can be found in venous blood. These circulating tumor cells are part of the metastatic cascade. Clinical studies have shown that these cells can be used to predict treatment response and their presence is strongly associated with poor survival prospects. Enumeration and characterization of CTCs is important as this can help clinicians make more informed decisions when choosing or evaluating treatment. CTC counts are being included in an increasing number of studies and thus are becoming a bigger part of disease diagnosis and therapy management. We present an overview of the most prominent CTC enumeration and characterization methods and discuss the assumptions made about the CTC phenotype. Extensive CTC characterization of for example the DNA, RNA and antigen expression may lead to more understanding of the metastatic process.

Figure 1

Formation of metastasis. Panel A shows a primary tumor after its inception. The heterogeneity of the tumor is indicated with different colors. Panel B shows blood vessels providing nutrients to the tumor (angiogenesis) leading to further growth and diversity. In this process either the tumor cells or the endothelial cells will need to penetrate the basal lamina. At this time tumor cells can enter the blood. The arrow depicts a CTC attached to the blood vessel wall of a distant organ. Panel C shows the formation of a metastasis after extravasation of the CTC. Only few CTC will have the characteristics necessary to create a metastasis.

Figure 2

Differentiation into heterogeneous metastases. A primary tumor in the breast creates generally spreads to the liver (panel A), lung (panel B) or bone (panel C). Due to tumor heterogeneity some tumor cells have a preference for a certain organ indicated by their color. Panel D shows a primary tumor in the prostate which only spreads to bone.

Figure 3

The frequency of erythrocytes, platelets, leukocytes and circulating tumor cells in blood of metastatic carcinoma patients and their cumulative probability[74].

Figure 4

The overall survival time of metastatic breast and prostate cancer patients was calculated from the date of the first follow-up blood draw after initiation of a new line of therapy. Kaplan-meier plots of the probability of overall survival for 123 patients with <1 CTC, 67 patients with 1-4 CTC, 55 patients with 5-24 CTC and 51 patients with >25 CTCs is shown. [74]

Figure 5

Examples of different CTC enrichment methods: Panel A) CellSearch which uses iron particles coupled to anti-EpCAM antibodies to enrich cells from epithelial origin. Cells are kept in a magnetic field during wash steps. After staining they are presented in a MagNest to pull all EpCAM positive cells to the imaging area. Panel B) Functionalized nano wire, here an EpCAM functionalized probe is placed directly in the blood stream to capture CTC. After 30 minutes the probe is removed from the blood and the attached cells are stained and fixed. The cells are investigated while still on the probe. Panel C) Rosettesep, here white blood cell depletion is achieved by forming aggregates between erythrocytes and CD45 positive cells. After which the aggregates are separated from the CTC using density separation. Panel D) passing whole blood through a sieve with 8 um pores. The erythrocytes and white blood cells will pass the pores while some of the bigger cells will stay on the membrane surface. The cells are fixed and stained on the filter.

Figure 6

Panel A, thumbnail images of CTC detected in metastatic cancer patient using CellSearch Row 1 one DAPI⁺ cell, CK⁺, CD45- => CTC. Row 2 four DAPI⁺ cells, one CK⁺, CD45^- CTC, one CK^dlm, CD45^- CTC, one CK^-, CD45⁺ leukocyte and one CK-, CD45- with no proof of origin. Panel B and C, detection of treatment targets Her2 and AR on CTC using immunofluorescence. Panel B, a Her2⁺ and Her2- CTC, Panel C, an AR⁺ and AR- CTC. For weak signals such as the androgen receptor a bright fluorophore is needed in this case the CK antibody is labeled with FITC and the AR labeled antibody with Phycoerythrin. Panels D and E, CTC can be restained for FISH to asses chromosomal abnormalities[71], panel D, or specific genes HER2, AR, PTEN, ERG [68].