Figure 8. TGFβ signalling is required for haematogenous but not lymphatic metastasis.

A: Representative images of primary tumours, lymph node metastases, intravasated cells, and lung metastases of ‘mixed’ tumours. Upper panels show mixed tumours containing control cells (red) and TGFβRDN cells (green). Middle panels show mixed tumours containing control cells (red) and Smad4shRNA cells (green). Lower panels mixed tumours containing control cells (red) and TGFβ1 over-expressing cells (green). Scale bar is 150μm. B: Quantification of metastasis of TGFβRDN and Smad4shRNA cells relative to control cells in the same mouse. Inguinal and axillary lymph nodes, circulating tumour cells and lung metastases are evaluated (average of 7, 4, 5 and 5 mice for TGFβRDN#1, TGFβRDN#2, shRNA#1 and shRNA#2, respectively). Quantification method is explained in Materials and Methods. ‘n.d’ indicates that no control or experimentally targeted cells were found in the blood. ‘0’ indicates that only control cells were detected. C: Quantification of TGFβ1 over-expressing MTLn3E metastasis relative to control cells in the same mouse. Inguinal and axillary lymph nodes, circulating tumour cells and lung metastases are evaluated (average of 8 mice – quantification performed as in part B). D: A 1:1 mixture of control cells expressing mCherry and control cells expressing GFP (columns 1&2) or control cells expressing mCherry and cells treated TGFβ1 2ng/ml for 24 hours expressing GFP (column 3) or a 1:1 mixture of control cells expressing mCherry and constitutively TGFβ1 IRES-GFP expressing cells (columns 4&5) were injected into the tail vein. 48hrs after injection the number of experimentally manipulated GFP expressing cells relative to control cells was assessed. Graph shows the ratio of GFP expressing cells to control mCherry expressing cells (the average and standard deviation from 4-6 different mice is shown). E: Left-hand image shows xy, xz, and yz sections of a ‘chain’ of MTLn3E cells (green) entering a lymphatic vessel. Middle panel shows motion analysis of the cells in the indicated region of the left-hand image. Right-hand panels show xy and yz sections of a group of cells within a lymphatic tumour draining from a mosaic tumour of control MTLn3E cells (red) and TGFβRDN MTLn3E cells (green).