Metastasis of Kaiso^positive and Kaiso^depleted cells to the lung results in dramatically different tumor behavior.

<p>(A-D) Kaiso^positive 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). Kaiso^depleted 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.</p

The molecular phenotype of the Kaiso^positive MDA-231 cells persist as they metastasize to other distal organs (liver and myocardium).

<p>(A) Neoplastic Kaiso^positive 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, Kaiso^depleted tumor cells are weakly stained for Kaiso (iv) and Vimentin (v) and negative for E-cadherin (vi). (B) Kaiso^positive tumor cells in lung metastases also stain strongly for MMP-2 (i), and MMP-9 (ii), while the Kaiso^depleted 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 Kaiso^positive 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.</p

Conceptual pathogenesis of cancer metastasis.

<p><b><i>Primary metastasis</i></b>: Invasion of Kaiso^positive and Kaiso^depleted 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, Kaiso^positive 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. Kaiso^depleted tumor cells form small aggregations that do not invade blood vessels therefore the secondary metastases do not occur. <b><i>Secondary metastasis</i></b>: The intravascular invasion by the Kaiso^positive 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.</p

IHC of primary subcutaneous Kaiso^positive and Kaiso^depleted MDA-231 tumors.

<p>Tumor cells (Tm) of Kaiso^positive (A-C) and Kaiso^depleted (D-F) masses do not invade the epidermis (double-headed arrow in A, D, F, arrow in B). Kaiso^positive tumor cells are labeled strongly positive for Kaiso (A) and vimentin (B) while the Kaiso^depleted 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.</p

Loss of Kaiso expression in breast cancer cells prevents intra-vascular invasion in the lung and secondary metastasis

<div><p>The metastatic activity of breast carcinomas results from complex genetic changes in epithelial tumor cells and accounts for 90% of deaths in affected patients. Although the invasion of the local lymphatic vessels and veins by malignant breast tumor cells and their subsequent metastasis to the lung, has been recognized, the mechanisms behind the metastatic activity of breast tumor cells to other distal organs and the pathogenesis of metastatic cancer are not well understood. In this study, we utilized derivatives of the well-established and highly metastatic triple negative breast cancer (TNBC) cell line MDA-MB-231 (MDA-231) to study breast tumor metastasis in a mouse model. These MDA-231 derivatives had depleted expression of Kaiso, a POZ-ZF transcription factor that is highly expressed in malignant, triple negative breast cancers. We previously reported that Kaiso depletion attenuates the metastasis of xenografted MDA-231 cells. Herein, we describe the pathological features of the metastatic activity of parental (Kaiso^positive) versus Kaiso^depleted MDA-231 cells. Both Kaiso^positive and Kaiso^depleted MDA-231 cells metastasized from the original tumor in the mammary fat pad to the lung. However, while Kaiso^positive cells formed large masses in the lung parenchyma, invaded large pulmonary blood vessels and formed secondary metastases and large tumors in the distal organs, Kaiso^depleted cells metastasized only to the lung where they formed small metastatic lesions. Importantly, intravascular invasion and secondary metastases in distal organs were not observed in mice xenografted with Kaiso^depleted cells. It thus appears that the lung may constitute a barrier for less invasive breast tumors such as the Kaiso^depleted TNBC cells; this barrier may limit tumor growth and prevents Kaiso^depleted TNBC cells from invading the pulmonary blood vessels and forming secondary metastases in distal organs.</p></div

Primary subcutaneous tumors formed by Kaiso^positive and kaiso^depleted cells with invasion of the lumen of surrounding veins.

<p>Subcutaneous tumor mass of Kaiso^positive MDA-231 human mammary carcinoma cells (Ai, ii) and Kaiso^depleted 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.</p

Intravascular invasion of secondary metastatic Kaiso^positive tumors.

<p>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. Kaiso^positive 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.</p

Kaiso^depleted MDA- 231 cells express negligible Kaiso compared to parental Kaiso^positive cells.

<p>Kaiso expression levels were determined using western blot. Both Kaiso^depleted clones (sh-K1 & sh-K2) expressed little Kaiso compared to the Kaiso^positive MDA-231 cells.</p

Thrombosis caused by Kaiso^positive tumors invading the blood vessels and heart ventricles.

<p>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.</p

Electron micrographs of damaged myelin sheaths from the spinal cord of rats infused intrathecally with kynurenic acid (KYNA) for 7 days.

<p>A–an area from the dorsal column of a rat infused with 0.01 nmol/min of KYNA with an astrocyte (As) and an oligodendrocyte (OL) surrounded by damaged myelin sheaths. B–in this detail of A delineated by the white box, a segment of well compacted thick myelin sheath (Ms) passes into a segment were all lamellae are widely separated due to disintegration of compaction at the intraperiod line indicated by arrows. C–an example of a damaged myelin sheath from a single axon (Ax) from the lateral column of the rat infused with 10 nmol/min KYNA were a few well compacted lamellae (white double headed arrows) are widely separated by uncompacted lamellae (black arrows). D–in the lateral column of a rat infused with 1 nmol/min of KYNA, an axon (Ax) has a damaged myelin with segmental loss of compaction due to separation of lamellae at the intraperiod line (white arrow). There is a well-compacted thick myelin sheath (Ms) in the adjacent axon. E–lateral column from a rat infused with 0.0002 pmol/min of KYNA per 7 days, with multiple myelin sheaths showing the segmental loss of compaction and one oligodendrocyte (OL). The box indicates the area displayed in higher magnification in F–with two axons (Ax) surrounded by uncompacted myelin lamellae. Size bars; A, E– 5 μM, B, C, D– 100 nM, F– 1 μM.</p