Soft fibrin gels promote selection and growth of tumorigenic cells

Abstract

The identification of stem-cell-like cancer cells through conventional methods that depend on stem cell markers is often unreliable. We developed a mechanical method for selecting tumorigenic cells by culturing single cancer cells in fibrin matrices of ~100 Pa in stiffness. When cultured within these gels, primary human cancer cells or single cancer cells from mouse or human cancer cell lines grew within a few days into individual round colonies that resembled embryonic stem cell colonies. Subcutaneous or intravenous injection of 10 or 100 fibrin-cultured cells in syngeneic or severe combined immunodeficiency mice led to the formation of solid tumours at the site of injection or at the distant lung organ much more efficiently than control cancer cells selected using conventional surface marker methods or cultured on conventional rigid dishes or on soft gels. Remarkably, as few as ten such cells were able to survive and form tumours in the lungs of wild-type non-syngeneic mice.

Fig. 1. Multicellular tumour spheroid formation in soft 3D fibrin gels

(a) A single B16-F1 cell grew into a multicellular tumour spheroid within 90-Pa 3D fibrin gel during culture course from Day 1 to Day 5. (b) Multicellular B16-F1 tumour spheroid formation after 5 days in culture within soft 3-D fibrin gels of different stiffness. (c) Multicellular tumour spheroid (round colony) number as a function of culture time: Day 1 to Day 6. The 90-Pa fibrin gel appears to be optimal for sustaining spheroid colony number. Mean ± s.e.m., n=6 (for 90-Pa gels) or 3 (for 420-Pa or 1050-Pa gels) independent experiments. There are no significant differences between 90-Pa and 420- Pa gels at Day 1 (p>0.2); there are significant differences at Day 2 through Day 6 (all p<0.022). There are significant differences between 90-Pa and 1050-Pa at Day 1 through Day 6 (all p<0.018). Between 420-Pa and 1050-Pa gels, there are differences only at Day 1 (p=0.037) and at Day 5 (p=0.00082). (d) Colony size of multicellular tumour spheroid as a function of culture time and fibrin stiffness. Apparently 90-Pa fibrin best promotes tumour growth. Mean ± s.e.m.; n=6 (for 90-Pa gels) or 3 (for 420-Pa or 1050-Pa gels) independent experiments. The stiffness of 3D fibrin gels with concentrations of 1 mg/ml, 4 mg/ml and 8 mg/ml is 90, 420 and 1050 Pa, respectively. There are no significant differences at Day 1 between 90 Pa and 420 Pa (p=0.27) or between 90-Pa and 1050-Pa gels (p=0.33); from Day 2 through Day 6, there are significant differences between 90-Pa and 420-Pa (p<0.002 for all paired comparisons) and between 90 Pa and 1050 Pa gels (all p<0.0012). The data in (c) and (d) are fitted by the 3^rd order polynomial functions (solid lines), the parameters of which are given in the Methods section.

Fig. 2. Tumour metastasis of 3D cultured B16-F1 cells in lung tissue of BALB/c mice

(a) B16-F1 tumour spheroids formed in soft 3D fibrin gel after 5-day culture were injected into tail vein of BALB/c mice with 10 cells. Mice were sacrificed after 2 months of injection and lung tissue image was recorded. B16-F1 cells cultured in 2D rigid dish were used as control. The results shown were a representative from three independent experiments. (b) Lung tissues of the above B16-F1 tumour spheroid treated BALB/c mice were analyzed by H&E staining. B16-F1 cells cultured on 2D rigid dish were used as control.

Fig. 3. Upregulation of stem cell-associated genes in B16-F1 spheroid cells cultured in 3D fibrin gel

(a) Stem cell marker expression in B16-F1 spheroid cells. Total mRNA of B16-F1 spheroid cells at Day 5 was extracted and used for the detection of Nanog, Oct3/4, CD133, nestin, Bmi-1 and c-kit mRNA expression by RT-PCR. B16-F1 cells cultured in 2D rigid dish were used as control. Three independent experiments showed similar results. (b) Murine telomerase reverse transcriptase subunit (mTERT) expression of B16-F1 spheroid cells. mTERT mRNA expression was measured by RT-PCR; representative images of 3 independent experiments. (c) and (d) Stem cells markers and mTERT expression in B16-F1 cells are quantified by real-time PCR. Same mRNA sample of B16-F1 tumour spheroid cells was used as above. B16-F1 cells cultured in 2D rigid dish were used as control. Mean ± s.e.m., *p <0.05, compared with control cells. (e) Apoptotic analysis of doxorubicin-treated 3D B16-F1 cells. Different concentrations of doxorubicin were added to the B16-F1 cell culture medium during the last 18 hours of 5-day culture in the 90-Pa 3D fibrin gels or conventional 2D rigid dish. Cells were collected and stained with FITC conjugated Annexin-V for apoptotic detection by flow cytometry. Mean ± s.e.m..; n=3 independent experiments; *p <0.05, compared with control cells.

Fig. 4. 3D fibrin-gel cultured B16-F1 cells do not stiffen but elevate tractions with substrate rigidity

(a) Cell stiffness (shear modulus) as a function of substrate stiffness. 3D fibrin gel cultured B16-F1 cells do not stiffen much with substrate rigidity, whereas control cancer cells do. Mean ± s.e.m., 5 independent experiments; at least 150 cells per experiment. (b) Both 3D fibrin gel cultured B16-F1 cells and control cells elevate tractions on stiffer substrates. The cells were plated for 6 hr before experiments in both (a) and (b). Mean ± s.e.m.; 3 independent experiments, n> 60 cells for each value (**, p<0.01; ***, p<0.001.)