A heterogeneous in vitro three dimensional model of tumour-stroma interactions regulating sprouting angiogenesis

Abstract

Angiogenesis, the formation of new blood vessels, is an essential process for tumour progression and is an area of significant therapeutic interest. Different in vitro systems and more complex in vivo systems have been described for the study of tumour angiogenesis. However, there are few human 3D in vitro systems described to date which mimic the cellular heterogeneity and complexity of angiogenesis within the tumour microenvironment. In this study we describe the Minitumour model--a 3 dimensional human spheroid-based system consisting of endothelial cells and fibroblasts in co-culture with the breast cancer cell line MDA-MB-231, for the study of tumour angiogenesis in vitro. After implantation in collagen-I gels, Minitumour spheroids form quantifiable endothelial capillary-like structures. The endothelial cell pre-capillary sprouts are supported by the fibroblasts, which act as mural cells, and their growth is increased by the presence of cancer cells. Characterisation of the Minitumour model using small molecule inhibitors and inhibitory antibodies show that endothelial sprout formation is dependent on growth factors and cytokines known to be important for tumour angiogenesis. The model also shows a response to anti-angiogenic agents similar to previously described in vivo data. We demonstrate that independent manipulation of the different cell types is possible, using common molecular techniques, before incorporation into the model. This aspect of Minitumour spheroid analysis makes this model ideal for high content studies of gene function in individual cell types, allowing for the dissection of their roles in cell-cell interactions. Finally, using this technique, we were able to show the requirement of the metalloproteinase MT1-MMP in endothelial cells and fibroblasts, but not cancer cells, for sprouting angiogenesis.

Figure 1. Characterization of the Minitumour spheroid model.

A - Fluorescent (left) and phase contrast (right) images of HUVEC, EndoFib and Minitumour spheroids before incubation in the collagen gel; endothelial cells pre-dyed with a CMFDA Green CellTracker dye are seen in each different spheroid type. B – Representative fluorescent images of spheroids after 48 h incubation in collagen gels, in the presence of complete medium, showing pre-dyed endothelial cells organized into pre-capillary sprouts. C – Quantification of endothelial sprout length from different spheroids show that MDA-MB-231 cells stimulate sprout formation even in the absence of exogenous growth factors VEGF and bFGF. D – Confocal (upper) and phase contrast (lower) images of MDA-MB231 cells pre-dyed with the green CellTracker dye in the Minitumour spheroid after 48 h incubation in complete medium. E - A 3D reconstruction of a Minitumour spheroid where the HUVECs have been dyed with a CMRA Orange CellTracker dye and the fibroblasts with a CMFDA Green Cell Tracker side panels show optical x and y sections of sprouts showing the deposition of HUVECs and Fibroblasts relative to sprout formation.

Figure 2. Multiphoton microscopy images of Minitumour spheroids after 40 h or 7 days culture.

A - HUVECs dyed with a CMFDA Green CellTracker dye were imaged within the Minitumour spheroid immediately following their embedding into the type-I collagen matrix using a Multiphoton microscope with a 20× objective. B – Immediately after collagen embedding, the collagen-I gel emits a weak homogenous Second Harmonic Generation (SHG) signal. C – Multiphoton imaging from of spheroids after 40 h incubation in the collagen-I gel shows the formation of green endothelial sprouts into the collagen matrix. D - The SHG signal from the collagen reveals an increase in matrix intensity around the endothelial sprouts. E – Merged image between CMFDA Green CellTracker dye and SHG signals after 40 h incubation. F – A higher amplification (40×) image of an endothelial cell sprout from a Minitumour spheroid after 40 h shows the alignment of collagen fibrils along the endothelial cell sprout (white arrows). G – Phase contrast images after 7 days incubation in the collagen-I gel showing a homogenous layer of cells. H – Multiphoton imaging after 7 days incubation shows the formation of a network of pre-dyed endothelial cells within the layer of cells. I – SHG signal from the collagen matrix after 7 days spheroid incubation. Scale bars represent 50 µm in F and 100 µm in all others.

Figure 3. Immunostaining of Minitumour spheroids show deposition of extracellular matrix components.

A – Minitumour spheroids incubated in collagen-I were immunostained with an anti-pan-laminin antibody and imaged by confocal microscopy showing the deposition of laminin around the endothelial sprouts after 40 h and D – a more widespread distribution after 7 days. B – Confocal microscopy of Minitumour spheroids immunostained with an anti-collagen IV antibody after 48 h shows a similar pattern, E – but after 7 days collagen-IV still localized around the endothelial cell sprouts. C – Confocal microscopy of Minitumour spheroids immunostained with an anti-Tenascin antibody show widespread distribution after 40 h, and F – after 7 days. All images were obtained using a 10× objective. Scale bars represent 100 µm.

Figure 4. Minitumour spheroids growth factor dependency.

A – Direct incubation of function blocking antibodies for VEGF or PDGF within the collagen-I gel decreases endothelial cell sprouting from the Minitumour spheroids. B – Minitumour spheroids incubated with function blocking antibodies to IL6 and IL8 show similar levels of sprout formation to EndoFib spheroids. C - Minitumour and EndoFib spheroids show a differential response to inhibition of growth factor signaling using small molecule growth factor receptor inhibitors. D – Increase in endothelial cell sprouting in both Minitumour and EndoFib spheroids after 40 h incubation with the gamma-secretase inhibitor DAPT. E – Representative images from Minitumour and EndoFib spheroids incubated in collagen-I for 40 h with the addition of different growth factor receptor inhibitors.

Figure 5. Minitumour spheroids have a different drug response than spheroids consisiting of only HUVEC and fibroblasts.

A – Incubation of EndoFib spheroids with different anti-angiogenic agents for 40 h shows inhibition of sprouting with Thalidomide, Endostatin and Galardin. B – Incubation of Minitumour spheroids with different anti-angiogenic agents for 40 h shows a response to Galardin but a non-significant effect of both Thalidomide and Endostatin.

Figure 6. MT1-MMP gene silencing in both HUVECs and fibroblasts decreases endothelial sprout formation.

Cells were infected with lentiviral particles expressing 2 different shRNAs against MT1-MMP, selected with puromycin and used to make spheroids. A – Representative images of pre-dyed endothelial cell sprouting from Minitumour spheroids made with HUVECs transduced with the different lentiviral derived shRNAs and controls. B – Quantification of endothelial cell sprouting showing a decrease in sprout formation with HUVECs expressing MT1-MMP shRNAs. C - Western Blots confirming MT1-MMP knock down levels of about 50% in HUVECs, adjusted relative intensity was determined through the ImageJ software follwed by normalization against the loading and mock controls. D – Representative images of pre-dyed endothelial cell sprouting from Minitumour spheroids made with Fibroblasts transduced with different lentiviral derived shRNAs and controls. E – Quantification of endothelial cell sprouting showing a decrease in sprout formation with from Minitumour spheroids with Fibroblasts expressing MT1-MMP shRNAs. F - Western blots confirming MT1-MMP knock down levels of about 30% in the fibroblasts, adjusted relative intensity was determined through the ImageJ software followed by normalization against the loading and mock controls.

Figure 7. Bioluminescence imaging of Minitumour spheroids reveals no difference in cancer cell proliferation with MMP inhibition.

A – Quantification of bioluminescence levels from MB231luc21H4 cell dilutions showing a linear relation between cell number and luciferase signal. B – Representative images showing the bioluminescence signals from sequential concentration dilutions of MB231luc21H4 cells. C – Quantification of total luminescence signal of Minitumour spheroids including MDA-MB-231-luc2 after 40 h incubation in collagen-I with galardin, a vector control and Nocodazole as a positive control for proliferation inhibition (p-value<0.05). D – Quantification of bioluminescence signal from Minitumour spheroids made with MB231luc21H4 and fibroblasts expressing lentiviral derived shRNAs for MT1-MMP and non-targeting controls.