Characterization of a novel metastatic prostate cancer cell line of LNCaP origin

Abstract

Background: The LNCaP cell line was originally isolated from the lymph node of a patient with metastatic prostate cancer. Many cell lines have been derived from LNCaP by selective pressures to study different aspects of prostate cancer progression. When injected subcutaneously into male athymic nude mice, LNCaP and its derivatives rarely metastasize.

Methods: Here, we describe the characteristics of a new LNCaP derivative, JHU-LNCaP-SM, which was generated by long term passage in normal cell culture conditions.

Results: Short tandem repeat (STR) analysis and genomic sequencing verified JHU-LNCaP-SM derivation from parental LNCaP cells. JHU-LNCaP-SM cells express the same mutated androgen receptor (AR) but unlike LNCaP, are no longer androgen dependent for growth. The cells demonstrate an attenuated androgen responsiveness in transcriptional assays and retain androgen sensitive expression of PSA, AR, and PSMA. Unlike parental LNCaP, JHU-LNCaP-SM cells quickly form subcutaneous tumors in male athymic nude mice, reliably metastasize to the lymph nodes and display a striking intra-tumoral and spreading hemorrhagic phenotype as tumor xenografts.

Conclusions: The JHU-LNCaP-SM cell line is a new isolate of LNCaP, which facilitates practical, preclinical studies of spontaneous metastasis of prostate cancer through lymphatic tissues.

Keywords: JHU-LNCaP-SM; PSMA; androgen; lymph node; metastasis.

Fig. 1

Genetic confirmation of LNCaP origin by DNA sequencing. To determine the origin of the JHU-LNCaP-SM cell line, genomic DNA sequencing analysis was performed on segments with known LNCaP AR mutations. The sequencing showed that the LNCaP, JHU-LNCaP-SM, and C4-2 lines all contained the LNCaP specific AR mutation (ACT–GCT).

Fig. 2

Relative growth rates and protein expression of AR and PSMA of LNCaP cells and derivatives in full and charcoal-stripped media culture conditions. (A) Growth rates of the LNCaP, JHU-LNCaP-SM, and C4-2 cell lines were determined by plating out equal densities in 96 well plates and following growth by MTS assay. To determine if the in vitro growth of the cell lines was androgen sensitive they were also cultured in normal or charcoal stripped serum conditions and followed as previous. (B) Lysates were taken of cell pellets from the LNCaP, JHU-LNCaP, and C4-2 cell lines grown in either normal or charcoal stripped serum media and western blot was used to determine relative AR and PSMA protein expression.

Fig. 3

Androgen receptor activity in cell culture. To determine if the cell lines were androgen sensitive a luciferase reporter assay was conducted on the LNCaP, JHU-LNCaP-SM, and C4-2 cell lines. All lines were stimulated with increasing titrations of the synthetic androgen (R1881) in cell culture and monitored by an androgen dependent promoter luciferase activity. n = 8; all concentrations for all cell lines had a P-value of <0.05 compared to the 0nM R1881 using the standard unpaired t-test except for 0 nM R1881 compared to 0.1 nM R1881 for JHU-LNCaP-SM. PSA expression levels were also determined for each cell line with androgen stimulation (insert).

Fig. 4

Xenograft growth rate in male athymic nude mice. (A) JHU-LNCaP-SM and LNCaP cells (2 × 10⁶) were injected subcutaneously in 6 week old male athymic nude mice and volume measurements were taken over a 30 day period. (B) A cohort of the JHU-LNCaP-SM xenograft animals were castrated to determine if the xenografts were androgen sensitive in vivo.

Fig. 5

In vivo and ex vivo characterization of JHU-LNCaP-SM xenografts. (A–C) Macroscopic characterization of primary tumors from JHU-LNCaP-SM xenografts revealed a consistently hemorrhagic appearance in vivo and ex vivo. (D) H&E staining of xenograft sections confirmed tumor presence and hemorrhagic phenotype.

Fig. 6

In vivo and ex vivo imaging of PSMA expression in JHU-LNCaP-SM and C4-2 xenografts. (A) Optical imaging of PSMA using YC27 showed that tumors could easily be non-invasively delineated from normal tissue in vivo. (B–D) Probe excretion through the urinary bladder can also be seen, shown as “B.” (E) Tumor uptake of YC27 showing mottled PSMA distribution was confirmed ex vivo as well as in whole mount sections. (F and G) PSMA sparsely expressing C4-2 xenografts were also easily delineated by NIRF imaging in vivo and ex vivo. (H) C4-2 shows a hemorrhagic phenotype but without the peri-tumoral subcutaneous spread.

Fig. 7

Proximal expression of PSMA in context with CD31 expression in LNCaP, JHU-LNCaP-SM, and C4-2 tumor xenografts. PSMA and CD31 expression patterns were determined using immunofluorescence microscopy upon primary tumor xenograft sections. (A and B) LNCaP tumors are represented, (C and D) represents JHU-LNCaP-SM tumors, and (E and F) depicts C4-2 xenograft tumors. Markers and channels are as indicated and are optimized for intensities individually. Scale bar = 100 μm.

Fig. 8

PSMA-expressing JHU-LNCaP-SM subcutaneous xenografts spontaneously metastasize to lymph nodes. (A) Ex vivo PSMA-targeted optical imaging of lymph nodes from selected mice that exhibited spontaneous metastasis from subcutaneous JHU-LNCaP-SM primary xenografts where green arrows show PSMA positive axillary lymph nodes and red is a negative node. (B and C) The red circle is a positive renal lymph node and also shown is a superficial cervical lymph node denoted by a green arrow. (D) Frozen sections of lymph node metastasis were analyzed by immunofluorescence for PSMA and CD31 expression and confirm the presence of PSMA-expressing cells within a vascular cage at the edge of packed lymphatic cells (scale bar = 100 μm). (E–H) H&E staining of various PSMA positive nodes confirming the presence of tumor cells (scale bar = 50μm).