Basement membrane-rich Organoids with functional human blood vessels are permissive niches for human breast cancer metastasis

Metastasic breast cancer is the leading cause of death by malignancy in women worldwide. Tumor metastasis is a multistep process encompassing local invasion of cancer cells at primary tumor site, intravasation into the blood vessel, survival in systemic circulation, and extravasation across the endothelium to metastasize at a secondary site. However, only a small percentage of circulating cancer cells initiate metastatic colonies. This fact, together with the inaccessibility and structural complexity of target tissues has hampered the study of the later steps in cancer metastasis. In addition, most data are derived from in vivo models where critical steps such as intravasation/extravasation of human cancer cells are mediated by murine endothelial cells. Here, we developed a new mouse model to study the molecular and cellular mechanisms underlying late steps of the metastatic cascade. We have shown that a network of functional human blood vessels can be formed by co-implantation of human endothelial cells and mesenchymal cells, embedded within a reconstituted basement membrane-like matrix and inoculated subcutaneously into immunodeficient mice. The ability of circulating cancer cells to colonize these human vascularized organoids was next assessed in an orthotopic model of human breast cancer by bioluminescent imaging, molecular techniques and immunohistological analysis. We demonstrate that disseminated human breast cancer cells efficiently colonize organoids containing a functional microvessel network composed of human endothelial cells, connected to the mouse circulatory system. Human breast cancer cells could be clearly detected at different stages of the metastatic process: initial arrest in the human microvasculature, extravasation, and growth into avascular micrometastases. This new mouse model may help us to map the extravasation process with unprecedented detail, opening the way for the identification of relevant targets for therapeutic intervention

Immunohistochemical characterization of control BME-rich organoids.

<p>Immunohistochemical characterization of explanted control BME-rich organoids using anti-CD34 (species specific: human and mouse; [39]), anti-CD45, anti-neutrophils, anti-vimentin and anti-α-SMA antibodies (Table S3). Cell nuclei were counterstained with hematoxylin. 10x and 40x images are shown.</p

Immunohistochemical characterization of human vascularized BME-rich organoids.

<p>Immunohistochemical characterization of explanted human vascularized BME-rich organoids using anti-CD34 (species specific: human and mouse; [39]), anti-CD45, anti-F4/80, anti-neutrophils, anti-vimentin and anti-α-SMA antibodies (Table S3). Cell nuclei were counterstained with hematoxylin. 10x and 40x images are shown.</p

Generation of BME-rich organoids with functional human blood vessels in immunodeficient mice.

<p>(a) A mixture of HUVEC^FLuc (3 x 10⁵) and human MSC (7.5 x 10⁴) were embedded in supplemented BME (150 µl) and inoculated subcutaneously in the ventral area (right lower quadrant) of immunodeficient mice. Human vascularized BME-rich organoids were easily identified as small bumps under the skin at all times <i>in vivo</i>. (b) Ventral D-luciferin-based F^Luc-BLI images of a representative mouse 1, 2, 4, 7, 10 and 13 weeks after implantation of the human vascularized BME-rich organoid. (c) Low magnification (4x) micrograph displaying the entire organoid (Scale bar is 1 mm). Hematoxylin and eosin (H and E)-stained sections taken from different part of the organoids revealed the presence of numerous luminal structures containing erythrocytes (arrowheads), and some glomeruloid microvascular proliferations (arrows). 10x and 40x images are shown.</p

Spontaneous metastasis from primary mammary fat pad tumors.

<p>MDA-MB-231^RLuc human breast cancer cells were implanted into the orthotopic environment of the left axillary MFP of nude mice bearing human vascularized BME-rich organoids (HVO) and control BME-rich organoids (CO), without human cells. Primary tumor growth (a, b), metastatic spread (b-e) and HVO functionality (b, c) were monitored over time <i>in vivo</i> and <i>ex vivo</i>. (a) Tumor growth curves for MDA-MB-231^RLuc (<i>n = 10</i>) orthotopic mammary tumors. (b) <i>In vivo</i> ventral coelenterazine-based R^Luc-BLI images (upper panels) and D-luciferin-based F^Luc-BLI images (lower panels) of a representative mouse. (c) <i>Ex vivo</i> coelenterazine-based R^Luc-BLI images (upper panels) and D-luciferin-based F^Luc-BLI images (lower panels) of excised lungs, inguinal lymph node (LN), spleen, liver, HVO and CO of a representative mouse. (d) Percentage of metastatic colonization and (e) tumor burden in harvested tissues (normal mouse organs and BME-rich organoids) of mice (<i>n = 8</i>) with primary tumor growth. Significant differences (*** p < 0.001).</p

Correlation between mRNA levels of Renilla Luciferase (R^Luc) and <i>ex vivo</i> coelenterazine-based R^Luc-bioluminescence of lungs (<i>n = 4</i>) and human vascularized BME-rich organoids (<i>n = 5</i>).

<p>In both cases R^Luc mRNA concentrations were well correlated (a: r = 0.829; b: r = 0.851) with <i>ex vivo</i> bioluminescence data.</p

Generation of control BME-rich organoids without human cells in immunodeficient mice.

<p>(a) Supplemented BME (150 μl) was inoculated subcutaneously in the ventral area (left lower quadrant) of immunodeficient mice. Control BME-rich organoids were easily identified as small bumps under the skin at all times <i>in vivo</i>. (b) Low magnification (4x) micrograph displaying the entire organoid (Scale bar is 1 mm). Hematoxylin and eosin (H and E)-stained sections taken from different part of the control organoids revealed the presence of numerous adipocytes, and some luminal structures containing erythrocytes (arrowheads). 10x and 40x images are shown.</p

Detection of disseminated tumor cells in human vascularized BME-rich organoids

<p><b>sections</b>. (a) The parenchyma contains a network of human CD34⁺ capillary-like microvessels (in green) and numerous isolated or clusters of CD44⁺ MDA-MB-231 tumor cells (in red), which are circulating (white arrow), attached to the endothelium (yellow arrow) or extravasating and colonizing the surrounding matrix (asterisk). (b) Small foci of CD44⁺ MDA-MB-231 tumor cells adjacent to human CD34⁺ capillary-like microvessels and glomeruloid microvascular proliferations. Nuclei are labeled in DAPI, x200. Scale bar is 25 μm.</p

Red de docentes y repositorio digital de recursos educativos: Una historia del capitalismo contemporáneo II. La crisis del fordismo a través fuentes fílmicas, literarias y estéticas

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