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. 2010 Dec 14;107(50):21677-82.
doi: 10.1073/pnas.1016234107. Epub 2010 Nov 22.

Malignant cells facilitate lung metastasis by bringing their own soil

Dan G Duda  1 , Annique M M J Duyverman, Mitsutomo Kohno, Matija Snuderl, Ernst J A Steller, Dai Fukumura, Rakesh K Jain
Affiliations

Malignant cells facilitate lung metastasis by bringing their own soil

Dan G Duda et al. Proc Natl Acad Sci U S A. .

Abstract

Metastatic cancer cells (seeds) preferentially grow in the secondary sites with a permissive microenvironment (soil). We show that the metastatic cells can bring their own soil--stromal components including activated fibroblasts--from the primary site to the lungs. By analyzing the efferent blood from tumors, we found that viability of circulating metastatic cancer cells is higher if they are incorporated in heterotypic tumor-stroma cell fragments. Moreover, we show that these cotraveling stromal cells provide an early growth advantage to the accompanying metastatic cancer cells in the lungs. Consistent with this hypothesis, we demonstrate that partial depletion of the carcinoma-associated fibroblasts, which spontaneously spread to the lung tissue along with metastatic cancer cells, significantly decreases the number of metastases and extends survival after primary tumor resection. Finally, we show that the brain metastases from lung carcinoma and other carcinomas in patients contain carcinoma-associated fibroblasts, in contrast to primary brain tumors or normal brain tissue. Demonstration of the direct involvement of primary tumor stroma in metastasis has important conceptual and clinical implications for the colonization step in tumor progression.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Tumors shed fragments containing viable cancer cells in blood circulation. (A) Schema of the tumor perfusion and blood collection setup: ds-Red-LLC1 tumors were grown in the kidney of mice ubiquitously expressing GFP, and tumor perfusate was collected by cannulating the efferent vein (renal vein). (B) Histogram of the composition of shed tumor cells/clumps obtained from the renal perfusion experiment (n = 5 mice). The majority of shed cancer cells were single or doublets. Host-derived GFP+ cells were present in all large clumps consisting of more than four to five cells. *P < 0.05. (C) Representative fluorescence multiphoton laser-scanning microscopy image of a heterogeneous clump, shed by a tumor using the isolated renal perfusion model. Green: stromal cells; red: tumor cells. (D) Viability of the shed cells and clumps using caspase staining: more than 22% of the ds-Red+ cancer cells within heterotypic (cancer plus host cells) clumps were negative for caspase 3 and 7, whereas only 12% of the cancer cells collected as single cells or in homotypic clumps were viable (P < 0.05). Data are expressed as mean ± SD of four independent experiments.
Fig. 2.
Fig. 2.
Passenger stromal cells survive and promote initial growth after i.v. infusion of tumor fragments. (A) Multiphoton microscopy image (630 μm across) of GFP+ primary tumor-derived stromal cells in a lung metastatic nodule in a C57BL/6 mouse 1 wk after i.v. infusion of DsRed-LLC1 tumor clumps obtained from a tumor growing in an Actb-GFP mouse. (B) Mean diameter of metastatic nodules increased significantly over time (*P < 0.05 vs. day 1). (C) Number of GFP+ cells per metastatic nodule significantly increased from day 1 to days 5–7 (*P < 0.05 vs. day 1, n = 4–5 mice). GFP+ cells are detectable for up to 2 wk in the macroscopic metastases. (D) Presence of GFP+ host cell in the initial metastases seems to provide a growth advantage: the ratio of foci with host cells increases from ≈40% on day 1 to 70–80% around day 7–10 (*P < 0.05 vs. day 1). After day 10, there is a “dilution” in GFP+ stromal cells (a decrease of the ratio to 35%), likely due to infiltration of the foci by non-GFP host-derived stromal cells.
Fig. 3.
Fig. 3.
Characterization of passenger stromal cells in a spontaneous metastasis formation model. (A and B) Transient parabiosis and BMT. (A) Transient parabiosis model using C57BL/6 and Actb-GFP/WT-BMT mice. (B) Fur depigmentation in Actb-GFP/WT-BMT mouse is secondary to the whole-body irradiation. (C) Representative multiphoton microscopy image of an LLC1 tumor grown in a successful GFP+ skin graft transplanted in a WT-C57BL mouse. The image is 630 μm across. (D and E) Phenotypic analysis of GFP+ primary tumor-derived stromal cells in metastases by immunohistochemistry and confocal microscopy: GFP+ cells frequently express αSMA (white arrows in D) and FSP1 (E). The subpanels are (1) blue, DAPI nuclear stain; (2) green, GFP; (3) red, Cy3-labeled antibody staining; and (4) merged image. Images are 280 μm across.
Fig. 4.
Fig. 4.
Carryover of primary tumor stromal cells in lung metastases increases metastasis formation and survival. (A–C) Representative confocal microscopy images of primary tumors generated by coimplatation of human CAFs with LLC1 cells, confirming the persistence of CAFs in 5-mm tumors (A), as well as CAF presence in the lungs after primary tumor resection (B and C). Blue: DAPI nuclear stain; red: Cy3-labeled anti-human vimentin; green: FITC-labeled anti-human HLA. Images are 700 μm across. (D) Survival in mice with LLC1 metastases. CAF depletion using systemic DT treatment after tumor resection significantly increased survival (*P < 0.001 vs. PBS control, n = 11–17 mice). (E and F) CAF depletion by DT treatment after tumor resection significantly reduced the number of spontaneous macrometastases in (E) LLC1 (LLC) and (F) LA-P0297 (LAP) models (*P < 0.05 vs. PBS control, n = 11–12 mice).
Fig. 5.
Fig. 5.
Clinical evidence for carryover of primary tumor stromal cells in human metastases. (A) Representative microscopy image of glioblastoma tissue. Red arrowheads indicate tumor vessels after αSMA/CD31 double staining. In normal human brain and primary brain tumors, only vessel-associated pericytes and vascular smooth muscle cells are αSMA positive. (B–D) Representative microscopy images of human brain metastases originating from (B) lung carcinoma, (C) renal cell carcinoma, and (D) breast carcinoma. Red arrowheads indicate αSMA-positive perivascular cells associated with CD31-positive vascular endothelial cells. Black arrowheads indicate focal presence of αSMA-positive tumor-associated fibroblasts. (E) Quantification of cases of human brain metastasis with detectable tumor-associated fibroblasts. (F) Representative microscopy image of human brain metastasis from endometrial carcinoma: endometrial stromal cells (CD10+ cells, blue arrowheads) are detectable in the brain in close association with the cancer cells (yellow arrowheads). (Scale bars, 50 μm.)
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