Establishment and serial passage of cell cultures derived from LuCaP xenografts

Abstract

Background: LuCaP serially transplantable xenografts derived from primary and metastatic human prostate cancer encompass the molecular and cellular heterogeneity of the disease and are an invaluable resource for in vivo preclinical studies. A limitation of this model, however, has been the inability to establish and passage cell cultures derived from the xenografts. Here, we describe a novel spheroid culture system that supports long-term growth of LuCaP cells in vitro.

Methods: Xenografts were minced and digested with collagenase. Tissue dissociation was terminated while the majority of cells remained as clusters rather than single cells. The cell clusters were suspended in StemPro medium supplemented with R1881 and Y-27632, a Rho kinase inhibitor, and placed in ultralow attachment dishes for spheroid culture. Serial passage was achieved by partial digestion to small clusters with trypsin/EDTA in the presence of Y-27632. Cell viability, growth and phenotype were monitored with LIVE/DEAD®, MTS, qRT-PCR, and immunocytochemical assays.

Results: Cells from six LuCaP xenografts formed proliferating spheroids that were serially passaged a minimum of three times and cryopreserved. Two of the cell lines, LuCaP 136 and LuCaP 147, were further passaged and characterized. Both expressed biomarkers characteristic of the xenografts of origin, were determined to be of independent origin by STR fingerprinting, and were free of mycoplasma. LuCaP 147 formed tumors similar to the original xenograft when injected into mice.

Conclusions: The ability to culture LuCaP cells affords new opportunities for fast, cheap, and efficient preclinical studies and extends the value of the LuCaP xenograft models.

Keywords: preclinical model; prostate cancer; spheroids.

Fig. 1

(A) Flowchart depicting digestion and culture of LuCaP xenograft-derived cells. (B) Flowchart depicting sequential filtration and collection of cells from digested LuCaP xenograft.

Fig. 2

Clusters of LuCaP cells survive in suspension culture. (A) Previous attempts to culture adherent LuCaP 35 and 96 cells on 3T3 feeder layers revealed a morphology of tight cell clusters (40X); (B) Morphology of various LuCaP-derived suspension cultures (40X); (C) LIVE/DEAD® assay of LuCaP 147 culture showing intact live (green) clusters compared to dead (red) single cells (100X); (D) Immunofluorescent staining of LuCaP 93 and 136 spheroids for keratin 18 (K18) and PCNA (100X).

Fig. 3

Subculture, cryopreservation, and proliferation of LuCaP cultured cells. Images of LuCaP 96 spheroids before (A), immediately following (B), and four days after digestion with trypsin/EDTA (C). Images of LuCaP 96 immediately following (D) and four days after thawing (E) as well as immunofluorescent detection of nuclear PCNA, indicating active proliferation (F). All images 100X.

Fig. 4

In vitro growth of LuCaP cultured cells. (A) LuCaP cells proliferated in vitro as shown by MTS assay of cultured LuCaP 136 and LuCaP 147 cells over seven days (*p < 0.005). (B) Response to androgen (+/− 10 nM R1881 for 48 hours) of various cultured LuCaP-derived cells as assayed by qRT-PCR for PSA expression (*p < 0.005).

Fig. 5

Immunofluorescence of prostate cell markers in cells from cultured LuCaP 136 and 147 spheroids. Cells lack expression of basal cell markers p63 (A and E) and K5 (C and G) but show heterogeneous staining for CD44 (B and F) and EpCAM (D and H). Cells are strongly positive for luminal cell markers AR (I and M) and K18 (J and N) as well as the proliferative marker PCNA (K and O) and HuNu (L and P), a marker of human nuclei.

Fig. 6

In vivo growth of cultured LuCaP 147 cells. (A) LuCaP 147 in vivo growth curve. (B) Comparison of LuCaP 147 tumors by H&E in the original xenograft (top) and the cell culture-derived xenograft (bottom) (100X).