Minimally invasive assessment of tumor angiogenesis by fine needle aspiration and flow cytometry

Abstract

The development of a new, less invasive, and more rapidly implemented method of quantifying endothelial cell density in tumors could facilitate experimental and clinical studies of angiogenesis. Therefore, we evaluated the utility of tumor fine needle aspiration (FNA) coupled with flow cytometry for assessment of tumor angiogenesis. Samples were obtained from cutaneous tumors of mice using FNA, then immunostained and assessed by flow cytometry to determine the number of CD31(+) endothelial cells. Results of the FNA/flow cytometry technique were compared with quantification of tumor microvessel density using immunohistochemistry. The ability of the FNA/cytometry technique to quantify the effects of anti-angiogenic therapy and to monitor changes in tumor angiogenesis over time in individual tumors was also determined. We found that endothelial cell percentages determined in tumor tissue aspirates by flow cytometry correlated well with the percentages of endothelial cells determined in whole tumor digests by flow cytometry and with tumor microvessel density measurements by immunohistochemistry. Moreover, we found that repeated FNA sampling of tumors did not induce endothelial cell changes. Interestingly, by employing repeated FNA sampling of the same tumors we were able to observe a sudden and marked decline in tumor angiogenesis triggered when tumors reached a certain size. Thus, we conclude that the FNA/flow cytometry technique is an efficient, reproducible, and relatively non-invasive method of rapidly assessing tumor angiogenesis, which could be readily applied to evaluation of tumor angiogenesis in clinical settings in humans.

Figure 1. Flow cytometric analysis of tumor endothelial cells

Fine needle aspirates (FNA) of tumors were processed for immunostaining and analyzed by flow cytometry as described in Methods. Cells that were PI⁺ were first excluded to eliminate dead cells from analysis. Then, live cells were gated using forward and side scatter characteristics to eliminate cell debris, and subsequent analysis of CD31 and CD11b stained cells. Endothelial cells were classified as CD31⁺CD11b^-.

Figure 2. Flow cytometric analysis of tumor FNA specimens and whole tumor digests yield similar estimates of tumor angiogenesis

Tumor specimens were collected by FNA or tumor biopsy from mice with established 4T1 tumors (A) or MCA-205 tumors (B), and analyzed by flow cytometry to determine percentages of CD31⁺ endothelial cells, as described in Methods. There was a significant (p = 0.0402) correlation between the percentage of CD31⁺ cells in mice (n = 27) with 4T1 tumors (A) and the percentage of CD31+ cells in mice (n = 15) with MCA-205 tumors (B, p = 0.0267). Multiple experiments were pooled for each tumor type in this analysis.

Figure 3. Tumor angiogenesis assessed by FNA/flow cytometry correlates with angiogenesis measured by immunohistochemistry (IHC)

Fine needle aspirate samples were collected from mice (n= 10 per group) with cutaneous 4T1 tumors and numbers of CD31⁺ endothelial cells were analyzed by flow cytometry (A), as described in Methods. Mice were euthanized, and tumors isolated and analyzed for MVD by IHC (B) as described in Methods. Correlation between MVD and percentage of CD31⁺ cells was determined using Spearman correlation, which demonstrated a significant (p = 0.1) correlation between the values. Results are representative of two independent experiments.

Figure 4. Repeated FNA sampling of tumors does not lead to endothelial cell artifacts

BALB/c mice with established 4T1 tumors were divided into two groups (n = 5 per group). In Group 1 mice, a single FNA was performed, while in Group 2 mice, 3 separate FNAs were performed, each 3 days apart. The percentage of CD31⁺ cells (mean ± SEM) was compared between the two groups by Mann-Whitney test. Significant differences in endothelial cell percentages were not observed (p < 0.05). Results are representative of two independent experiments with 5 mice per group.

Figure 5. FNA analysis can be used to assess the effects of anti-angiogenic therapy (ZD6474)

BALB/c mice (n=5 per group) with established mammary fat pad 4T1 tumors were treated daily with ZD6474 (25 mg/kg) or with control buffer by oral gavage. Percentages of CD31⁺ endothelial cells in treated and sham-treated tumors were determined (mean ± SEM) using FNA/flow cytometry, as described in Methods. The percentage of CD31⁺ endothelial cells was significantly decreased (p < 0.05) in ZD6474-treated tumors, compared to control tumors, as assessed by Mann-Whitney test. Results are representative of two independent experiments.

Figure 6. Repeated measurement of tumors by FNA reveals angiogenic collapse of the tumor

Syngeneic mice were challenged with 4T1 (A) or MCA-205 (B) tumors. Tumor measurements were initiated to coincide with the first FNA. FNA were taken every three days after the initial FNA until the first mouse had a maximal tumor diameter of 10 mm, at which time all mice were sacrificed. Tumors from mice challenged with 4T1 and MCA-205 tumors had a rapid decrease in the percentage of endothelial cells present suggestive of an angiogenic collapse. Results depict the mean (±SEM) of endothelial cells as determined by FNA and tumor growth as represented by maximal tumor diameter. Results are representative of two independent experiments.