Fig 1.
Kaisodepleted MDA- 231 cells express negligible Kaiso compared to parental Kaisopositive cells.
Kaiso expression levels were determined using western blot. Both Kaisodepleted clones (sh-K1 & sh-K2) expressed little Kaiso compared to the Kaisopositive MDA-231 cells.
Fig 2.
Primary subcutaneous tumors formed by Kaisopositive and kaisodepleted cells with invasion of the lumen of surrounding veins.
Subcutaneous tumor mass of Kaisopositive MDA-231 human mammary carcinoma cells (Ai, ii) and Kaisodepleted tumor cells (Aiii, iv)) implanted into the fat pad of the mammary gland of female NRG mice. Tumor cells abut against the epidermis (arrow in Ai, iii) but do not invade it. Tumor cells are large, markedly pleomorphic, there is high mitotic index. (Bi) A vein (Bv, delineated by arrowheads) is adjacent to the subcutaneous tumor mass (Tm). It is distended by clumps and individual large pleomorphic cells (Bii) and also has scattered red blood cells. H&E. Size bars Ai, ii, Bi– 500 microns; Aii, iv, Bii– 50 microns.
Fig 3.
Metastasis of Kaisopositive and Kaisodepleted cells to the lung results in dramatically different tumor behavior.
(A-D) Kaisopositive tumors are numerous, large, obliterate the architecture of the pulmonary tissue and invade the lumen of large blood vessels (arrow in A). A segment of the vascular wall indicated by yellow arrows (B, D) is obliterated by tumor cells (double-headed interrupted arrow in B) that provide continuity between a perivascular mass and intravascular tumors (ivT) in the lumen (asterix in B, C). Apparent vascular invasion is associated with formation of intravascular thrombus (C, D). Intravascular surface of tumors or tumor thrombi is typically lined by endothelial cells (B). Tumor cells in intravascular masses or thrombi are large and pleomorphic (B, D). Kaisodepleted cells form small interstitial aggregations (arrow in E) of large pleomorphic cells (F, G) that do not invade the wall or the lumen (F) of adjacent blood vessels. H&E–A, B, E, F; Masson’s trichrome (C, D, G). Size bars; A, E– 500 microns, B-D, F, G– 50 microns.
Fig 4.
Intravascular invasion of secondary metastatic Kaisopositive tumors.
Low magnification images (A, C, E) and high magnification images (B, D, F) of tissue regions outlined by white dotted lines in A, C and E. Kaisopositive cells metastatic to the liver (A, B) and kidney (C, D) formed large tumors and invaded adjacent blood vessels with formation of thrombi (Th in B, D) delineated from the surrounding tissue by yellow arrows. Tumor masses in the myocardium (white box and arrowhead in E) often resulted in invasion of the ventricle (asterix) with formation of a mass (white box in E) and thrombus (Th in F). Thrombus is delineated from myocardium (my) by yellow arrows in F. There is continuity between the masses of tumor cells in the myocardium and in the intraventricular thrombus (F). H&E. Size bars; A, C, E– 1,000 microns, B, D, F– 50 microns.
Fig 5.
Thrombosis caused by Kaisopositive tumors invading the blood vessels and heart ventricles.
In the lung (A), a number of large blood vessels (two indicated by arrows) have intravascular thrombi delineated from the vascular wall by yellow arrows and protruding in the vascular lumen (Th in Aii, iii). The thrombi are infiltrated by neoplastic cells and are lined by endothelium (solid arrowheads in Aii) or not (open arrowhead in Aiii). In the myocardium (my, B) thrombi protruding into the ventricular lumen (Bi, iii) are also infiltrated by neoplastic cells (Th in Bii, iv) and either lined by endothelium (solid arrowheads in Bii) or not (open arrowheads in Biv). H&E. Size bars; Biii– 1,000 microns, Ai, Bi– 500 microns, Aii, iii, Bii, iv B– 50 microns.
Fig 6.
IHC of primary subcutaneous Kaisopositive and Kaisodepleted MDA-231 tumors.
Tumor cells (Tm) of Kaisopositive (A-C) and Kaisodepleted (D-F) masses do not invade the epidermis (double-headed arrow in A, D, F, arrow in B). Kaisopositive tumor cells are labeled strongly positive for Kaiso (A) and vimentin (B) while the Kaisodepleted cells are labelled considerably less (D, E). The labeling with anti-E-cadherin antibody is negative for both types of tumor cells in contrast to the positive labelling of the mouse epithelium in sebaceous glands (Sb in C, F) and in epidermis (F). Size bars A-F– 50 microns.
Fig 7.
The molecular phenotype of the Kaisopositive MDA-231 cells persist as they metastasize to other distal organs (liver and myocardium).
(A) Neoplastic Kaisopositive cells in lung metastases or thrombi are large, pleomorphic, and stain positive for Kaiso (i) and vimentin (ii), but negative for E-cadherin (iii). In contrast, Kaisodepleted tumor cells are weakly stained for Kaiso (iv) and Vimentin (v) and negative for E-cadherin (vi). (B) Kaisopositive tumor cells in lung metastases also stain strongly for MMP-2 (i), and MMP-9 (ii), while the Kaisodepleted tumor cells stain weakly for MMP-2 (iii), but positive for MMP-9 (iv). The asterisk indicates the lumen of the blood vessel with the thrombus (Th) and the yellow arrows indicate the vascular wall and its obliteration by tumor cells in (A, B). (C) The thrombus (Th) formed in the large blood vessel of the liver (i-v) and in the ventricle of the heart (vi-viii) in mice injected with Kaisopositive cells partially obliterated the vascular wall or endocardium indicated by the yellow arrows (i-viii). Neoplastic cells are stained positive for Kaiso (i, vi), vimentin (ii, vii), MMP-2 (iv, ix), and MMP-9 (v, x), and negative for E-cadherin (iii, viii). Scale bars; A-C– 50 microns.
Fig 8.
Conceptual pathogenesis of cancer metastasis.
Primary metastasis: Invasion of Kaisopositive and Kaisodepleted mammary carcinoma cells of the local veins and lymphatics allows the cells to migrate via the right heart ventricle to the lung where they are trapped in the capillary blood vessels (b.v.) and form pulmonary metastases. While in the lung, Kaisopositive cells proliferate successfully and form large, coalescing masses that send the cells to actively cross the wall of adjacent blood vessels and invade their lumen. Kaisodepleted tumor cells form small aggregations that do not invade blood vessels therefore the secondary metastases do not occur. Secondary metastasis: The intravascular invasion by the Kaisopositive tumor cells in the lung presumably leads to its migration in the blood via the left heart to a variety of organs notably heart, liver and kidney, where they form metastases and tumors with the invasion of local blood vessels or heart ventricles in a fashion similar to that observed in the lung. This may lead to tertiary metastases; via the venous flow to the right heart and ultimately to the lung.