Dissociated Primary Human Prostate Cancer Cells Coinjected with the Immortalized Hs5 Bone Marrow Stromal Cells Generate Undifferentiated Tumors in NOD/SCID-γ Mice

Xin Chen,Asha S Multani,Qiuhui Li,Tammy Calhoun-Davis,Bigang Liu,Sofia Honorio,Xin Liu,Can Liu,Dean G Tang,Amit Singh

doi:10.1371/journal.pone.0056903

Abstract

Reconstitution of tumor development in immunodeficient mice from disaggregated primary human tumor cells is always challenging. The main goal of the present study is to establish a reliable assay system that would allow us to reproducibly reconstitute human prostate tumor regeneration in mice using patient tumor-derived single cells. Using many of the 114 untreated primary human prostate cancer (HPCa) samples we have worked on, here we show that: 1) the subcutaneum represents the most sensitive site that allows the grafting of the implanted HPCa pieces; 2) primary HPCa cells by themselves fail to regenerate tumors in immunodeficient hosts; 3) when coinjected in Matrigel with rUGM (rat urogenital sinus mesenchyme), CAF (carcinoma-associated fibroblasts), or Hs5 (immortalized bone marrow derived stromal) cells, primary HPCa cells fail to initiate serially transplantable tumors in NOD/SCID mice; and 4) however, HPCa cells coinjected with the Hs5 cells into more immunodeficient NOD/SCID-IL2Rγ−/− (NSG) mice readily regenerate serially transplantable tumors. The HPCa/Hs5 reconstituted ‘prostate’ tumors present an overall epithelial morphology, are of the human origin, and contain cells positive for AR, CK8, and racemase. Cytogenetic analysis provides further evidence for the presence of karyotypically abnormal HPCa cells in the HPCa/Hs5 tumors. Of importance, HPCa/Hs5 xenograft tumors contain EpCAM+ cells that are both clonogenic and tumorigenic. Surprisingly, all HPCa/Hs5 reconstituted tumors are undifferentiated, even for HPCa cells derived from Gleason 7 tumors. Our results indicate that primary HPCa cells coinjected with the immortalized Hs5 stromal cells generate undifferentiated tumors in NSG mice and we provide evidence that undifferentiated HPCa cells might be the cells that possessed tumorigenic potential and regenerated HPCa/Hs5 xenograft tumors.

Highlights

Prostate cancer (PCa) is the leading malignancy with estimated,241,740 new cases and, 28,170 deaths in the USA in 2012 [1]
The human PCa (HPCa) samples were used in 2 types of experiments: either transplanted, as small pieces, in male immunodeficient mice to generate xenografts or used freshly isolated single cells for in vitro and in vivo studies (Fig. 1)
In NOD/SCID mice, which were used most often, we observed increasing tumor takes with increasing tumor grade at s.c and kidney capsule (KC) sites (Table 1; Table S3)

Summary

Introduction

Prostate cancer (PCa) is the leading malignancy with estimated ,241,740 new cases and , 28,170 deaths in the USA in 2012 [1]. Human cancers harbor a population of stem-like cancer cells operationally termed cancer stem cells (CSCs), which are believed to be responsible for tumor initiation, promotion, progression, metastasis, and treatment resistance [2]. Prostate CSCs reported by several groups are less differentiated expressing little/no AR (androgen receptor) and PSA (prostatespecific antigen). Using a PSA promoter-driven GFP lentiviral reporter, we have purified out differentiated (PSA+) and undifferentiated (PSA2/lo) PCa cells for gene expression profiling and functional studies and found that the PSA2/lo cell population harbors long-term tumor-propagating cells that resist to castration [25]. Our study suggests that the undifferentiated PSA2/lo PCa cell population likely represents a pre-existent cell-of-origin for CRPC [25]

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