Generation of vessel co-option lung metastases mouse models for single-cell isolation of metastases-derived cells and endothelial cells

Abstract

Tumor vessel co-option, a process in which cancer cells "hijack" pre-existing blood vessels to grow and invade healthy tissue, is poorly understood but is a proposed resistance mechanism against anti-angiogenic therapy (AAT). Here, we describe protocols for establishing murine renal (RENCA) and breast (4T1) cancer lung vessel co-option metastases models. Moreover, we outline a reproducible protocol for single-cell isolation from murine lung metastases using magnetic-activated cell sorting as well as immunohistochemical stainings to distinguish vessel co-option from angiogenesis. For complete details on the use and execution of this protocol, please refer to Teuwen et al. (2021).

Keywords: Antibody; Cancer; Cell isolation; Model organisms; Single cell.

Graphical abstract

Figure 1

Set-up of RENCA model and 4T1 model (A) Set-up of RENCA model and treatment schedule. (B) Set-up of 4T1 model and treatment schedule. This figure is taken from Teuwen et al. (Teuwen et al., 2021).

Figure 2

Isolation of metastases-derived cells and EC-s from mouse lung metastases Scheme illustrating isolation of cells from metastases (1) and subsequent isolation of ECs from lung metastases (2, 3). For EC isolation, a MACS immune & epithelial cell depletion (2) and EC enrichment (3) was performed. The included H&E image shows lung metastases from the RENCA model (Arrows indicate metastases; scale bar: 250 μm). This figure is adapted from Conchinha et al. (Conchinha et al., 2021).

Figure 3

Distinguishing vessel co-option from angiogenic metastases (A) Workflow for immunostaining of murine lung tissue. (B) Representative images of renal cancer lung metastases, stained for EdU, CD34, and Hoechst (nuclei). Middle images are magnifications of respective boxed areas. Arrowheads, proliferating ECs; scale bar 50 μm. (C) Quantification of the percentage of proliferating ECs in (B). Data are means ± SEM; n = 3–6. ∗p < 0.05 by one-way ANOVA with Tukey’s multiple comparisons test. (D) Representative images of renal cancer lung metastases stained for ESM-1, CD34, and Hoechst (nuclei). Middle images are magnifications of the respective boxed areas. Arrowheads, tip ECs; scale bar 50 μm. (E) Quantification of ESM-1+ ECs per CD34+ area in (D). Data are means ± SEM; n = 3. ∗p < 0.05 by one-way ANOVA with Tukey’s multiple comparisons test. (F) Representative micrographs of angiogenic (top) and vessel co-option (bottom) renal cancer lung metastases sections, immunostained for CD31 (ECs) and PDPN (pneumocytes) on adjacent sections. Nuclei are counterstained with Hoechst. Images in the middle are magnifications of the respective boxed areas. Note how the metastasis, relying on angiogenic vessel sprouting, grows as a sphere that compresses the surrounding lung parenchyma and into which CD31low vessels sprout (inset), while the cancer cells of the metastasis, relying on vessel co-option, invade the surrounding lung tissue in an irregular/infiltrative manner with preservation of the epithelial and vascular structure. Dotted yellow line: border between metastatic tissue and surrounding lung tissue; white arrowheads: angiogenic vessels, yellow arrows: border between the metastasis and the compressed surrounding lung tissue; asterisks, co-opting cancer cells; scale bar, 100 μm. (G) Quantification of vascularization type in the RENCA model in the indicated conditions. Data are mean ± SEM; n=3–5. This figure is adapted from Teuwen et al. (Teuwen et al., 2021).