Optoacoustics delineates murine breast cancer models displaying angiogenesis and vascular mimicry

Abstract

Background: Optoacoustic tomography (OT) of breast tumour oxygenation is a promising new technique, currently in clinical trials, which may help to determine disease stage and therapeutic response. However, the ability of OT to distinguish breast tumours displaying different vascular characteristics has yet to be established. The aim of the study is to prove OT as a sensitive technique for differentiating breast tumour models with manifestly different vasculatures.

Methods: Multispectral OT (MSOT) was performed in oestrogen-dependent (MCF-7) and oestrogen-independent (MDA-MB-231) orthotopic breast cancer xenografts. Total haemoglobin (THb) and oxygen saturation (SO₂^MSOT) were calculated. Pathological and biochemical evaluation of the tumour vascular phenotype was performed for validation.

Results: MCF-7 tumours show SO₂^MSOT similar to healthy tissue in both rim and core, despite significantly lower THb in the core. MDA-MB-231 tumours show markedly lower SO₂^MSOT with a significant rim-core disparity. Ex vivo analysis revealed that MCF-7 tumours contain fewer blood vessels (CD31+) that are more mature (CD31+/aSMA+) than MDA-MB-231. MCF-7 presented higher levels of stromal VEGF and iNOS, with increased NO serum levels. The vasculogenic process observed in MCF-7 was consistent with angiogenesis, while MDA-MB-231 appeared to rely more on vascular mimicry.

Conclusions: OT is sensitive to differences in the vascular phenotypes of our breast cancer models.

Fig. 1

Optoacoustic tomography reveals oestrogen-independent MDA-MB-231 tumours have poorer oxygenation than oestrogen-dependent MCF-7 tumours and healthy tissue. a Regions of interest (yellow outline) were drawn in the optoacoustic tomography slice at which the tumour area burden was highest. A region containing the aorta and inferior vena cava was used as a baseline reference in normal tissue. Representative images show the spatial distribution of b oxygenation (SO₂^MSOT) and (c) total haemoglobin (THb) with tumour regions of interest overlaid (yellow outline). Quantification graphs are shown on the right, data were extracted from all regions of interest, and show a significantly higher oxygenation in MCF-7 compared to MDA-MB-231 (b) tumours and a decrease in THb (c). n^MCF−7 = 11; n²³¹ = 16, data expressed as mean ± SEM. *p < 0.05, ****p < 0.0001. Statistical significance was assessed by paired two-tailed t-test within a single-tumour type and by unpaired two-tailed t-test between tumour types

Fig. 2

Optoacoustic tomography provides a non-invasive assessment of the rim–core vascular phenotypes of both breast cancer models. a Rim data were taken from a region of interest drawn around the outside of the tumour, then shrunk by a radial distance of 1 mm. Significant differences in THb and SO₂^MSOT were seen between the rim and core of MDA-MB-231 tumours, though only THb showed a rim–core variation in MCF-7 tumours b, c. Extracting “large” (20–30 mm² OT area) tumours for a size matched analysis (d) showed similar THb values but different SO₂^MSOT between rims and cores e, f. Statistical significance was assessed by paired two-tailed t-test within a single-tumour type and by unpaired two-tailed t-test between tumour types. For (b) and (c) n^MCF−7 = 11; n²³¹ = 15, for (e) and (f) n^MCF−7 and n²³¹ = 4. All panels data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

MDA-MB-231 exhibit a high-microvessel density but relatively poor maturity compared to MCF-7. a IHC representative micrographs for each tumour type stained with CD31 to mark endothelial cells and ASMA to mark the supporting pericyte layer (ASMA+ cells surrounding blood vessels). The lowest panel shows the mask used to count co-localised ASMA and CD31 staining on adjacent sections (orange overlap/yellow CD31+ only). Arrowheads indicate CD31+ with no ASMA staining in MDA-MB-231. Scale bar = 30 µm. MDA-MB-231 tumours show increased overall CD31 staining (b) and microvessel density (MVD, c), but decreased vessel thickness (d) and ASMA coverage (e) compared to MCF-7. For b, c and d, n^MCF−7 = 12 and n²³¹ = 16. For E, n^MCF−7 = 7 and n²³¹ = 4. All panels, data expressed as mean ± SEM. ***p < 0.001, ****p < 0.0001 by unpaired two-tailed t-test

Fig. 4

The hypoxic and inflammatory phenotype differs between the two breast tumour models. MDA-MB-231 cells have a lower oxygen consumption rate (a) yet show higher hypoxia in tumours (b). VEGF staining is lower in MDA-MB-231 tumours (c). Serum mouse VEGF (mVEGF, d) and nitric oxide (NO, e) are also lower in MDA-MB-231 tumours. f MCF-7 shows an inflammatory phenotype, with higher staining for iNOS and Arginase indicating the presence of type 1 and 2 macrophages respectively. Scale bars in (b) and (c) = 50 µm; f = 20 µm. For (b) and (c) n^MCF−7 = 12 and n²³¹ = 16. For (d) and (e) n^MCF−7 = 6 and n²³¹ = 9. All panels, data expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired two-tailed t-test

Fig. 5

Assessment of vascular mimicry. a Representative micrographs (magnification ×20) of tubular-like structures generated in matrigel 3D in vitro culture for MDA-MB-231 cells; no such structures are observed in MCF-7 cells. This phenotype is associated with vascular mimicry. b Representative micrographs (magnification ×40) of tumour sections stained with PAS and CD31. c All PAS-positive blood vessels were identified and the CD31 positivity of these blood vessels was then evaluated. The number of CD31+/PAS+ blood vessels was significantly lower in MDA-MB-231 compared to MCF-7 tumours. d Western blot for protein levels of VE-Cadherin in MCF-7 and MDA-MB-231 xenograft tumours ex vivo provided confirmation of these in vitro findings. GAPDH is shown as a house-keeping protein. b, c n^MCF−7 = 6 and n²³¹ = 6 data expressed as mean ± SEM, *p < 0.05 by unpaired two-tailed t-test. d n^MCF−7 = 8 and n²³¹ = 15