BackgroundThere is resurgence within drug and biomarker development communities for the use of primary tumorgraft models as improved predictors of patient tumor response to novel therapeutic strategies. Despite perceived advantages over cell line derived xenograft models, there is limited data comparing the genotype and phenotype of tumorgrafts to the donor patient tumor, limiting the determination of molecular relevance of the tumorgraft model. This report directly compares the genomic characteristics of patient tumors and the derived tumorgraft models, including gene expression, and oncogenic mutation status.MethodsFresh tumor tissues from 182 cancer patients were implanted subcutaneously into immune-compromised mice for the development of primary patient tumorgraft models. Histological assessment was performed on both patient tumors and the resulting tumorgraft models. Somatic mutations in key oncogenes and gene expression levels of resulting tumorgrafts were compared to the matched patient tumors using the OncoCarta (Sequenom, San Diego, CA) and human gene microarray (Affymetrix, Santa Clara, CA) platforms respectively. The genomic stability of the established tumorgrafts was assessed across serial in vivo generations in a representative subset of models. The genomes of patient tumors that formed tumorgrafts were compared to those that did not to identify the possible molecular basis to successful engraftment or rejection.ResultsFresh tumor tissues from 182 cancer patients were implanted into immune-compromised mice with forty-nine tumorgraft models that have been successfully established, exhibiting strong histological and genomic fidelity to the originating patient tumors. Comparison of the transcriptomes and oncogenic mutations between the tumorgrafts and the matched patient tumors were found to be stable across four tumorgraft generations. Not only did the various tumors retain the differentiation pattern, but supporting stromal elements were preserved. Those genes down-regulated specifically in tumorgrafts were enriched in biological pathways involved in host immune response, consistent with the immune deficiency status of the host. Patient tumors that successfully formed tumorgrafts were enriched for cell signaling, cell cycle, and cytoskeleton pathways and exhibited evidence of reduced immunogenicity.ConclusionsThe preservation of the patient’s tumor genomic profile and tumor microenvironment supports the view that primary patient tumorgrafts provide a relevant model to support the translation of new therapeutic strategies and personalized medicine approaches in oncology.

RNA interference (RNAi) is a natural cellular regulatory process that inhibits gene expression by transcriptional, post-transcriptional and translational mechanisms. Synthetic approaches that emulate this process (small interfering RNA (siRNA), short hairpin RNA (shRNA)) have been shown to be similarly effective in this regard. We developed a novel 'bifunctional' RNAi strategy, which further optimizes target gene knockdown outcome. A bifunctional construct (bi-sh-STMN1) was generated against Stathmin1, a critical tubulin modulator that is overexpressed in human cancers. The bifunctional construct is postulated to concurrently repress the translation of the target mRNA (cleavage-independent, mRNA sequestration and degradation) and degrade (through RNase H-like cleavage) post-transcriptional mRNA through cleavage-dependent activities. Bi-sh-STMN1 showed enhanced potency and durability in parallel comparisons with conventional shRNA and siRNAs targeting the same sequence. Enhanced STMN1 protein knockdown by bi-sh-STMN1 was accompanied by target site cleavage at the mRNA level showed by the rapid amplification of complementary DNA ends (RACE) assay. Bi-sh-STMN1 also showed knockdown kinetics at the mRNA level consistent with its multieffector silencing mechanisms. The bifunctional shRNA is a highly effective and advantageous approach mediating RNAi at concentrations significantly lower than conventional shRNA or siRNA. These results support further evaluations.

Bifunctional small hairpin RNAs (bi-shRNAs) are functional miRNA/siRNA composites that are optimized for posttranscriptional gene silencing through concurrent mRNA cleavage-dependent and -independent mechanisms (Rao et al., 2010 ). We have generated a novel bi-shRNA using the miR30 scaffold that is highly effective for knockdown of human stathmin (STMN1) mRNA. STMN1 overexpression well documented in human solid cancers correlates with their poor prognosis. Transfection with the bi-shSTMN1-encoding expression plasmid (pbi-shSTMN1) markedly reduced CCL-247 human colorectal cancer and SK-Mel-28 melanoma cell growth in vitro (Rao et al., 2010 ). We now examine in vivo the antitumor efficacy of this RNA interference-based approach with human tumor xenografted athymic mice. A single intratumoral (IT) injection of pbi-shSTMN1 (8 μg) reduced CCL-247 tumor xenograft growth by 44% at 7 days when delivered as a 1,2-dioleoyl-3-trimethyl-ammoniopropane:cholesterol liposomal complex. Extended growth reductions (57% at day 15; p < 0.05) were achieved with three daily treatments of the same construct. STMN1 protein reduction was confirmed by immunoblot analysis. IT treatments with pbi-shSTMN1 similarly inhibited the growth of tumorgrafts derived from low-passage primary melanoma (≥70% reduction for 2 weeks) and abrogated osteosarcoma tumorgraft growth, with the mature bi-shRNA effector molecule detectable for up to 16 days after last injection. Antitumor efficacy was evident for up to 25 days posttreatment in the melanoma tumorgraft model. The maximum tolerated dose by IT injection of >92 μg (Human equivalent dose [HED] of >0.3 mg/kg) in CCL-247 tumor xenograft-bearing athymic mice was ∼10-fold higher than the extrapolated IC(50) of 9 μg (HED of 0.03 mg/kg). Healthy, immunocompetent rats were used as biorelevant models for systemic safety assessments. The observed maximum tolerated dose of <100 μg for intravenously injected pbi-shSTMN1 (mouse equivalent of <26.5 μg; HED of <0.09 mg/kg) confirmed systemic safety of the therapeutic dose, hence supporting early-phase assessments of clinical safety and preliminary efficacy.

BackgroundOsteosarcoma (OS) is the most common type of solid bone cancer, with latent metastasis being a typical mode of disease progression and a major contributor to poor prognosis. For this to occur, cells must resist anoikis and be able to recapitulate tumorigenesis in a foreign microenvironment. Finding novel approaches to treat osteosarcoma and target those cell subpopulations that possess the ability to resist anoikis and contribute to metastatic disease is imperative. Here we investigate anchorage-independent (AI) cell growth as a model to better characterize anoikis resistance in human osteosarcoma while using an expression profiling approach to identify and test targetable signaling pathways.MethodsEstablished human OS cell lines and patient-derived human OS cell isolates were subjected to growth in either adherent or AI conditions using Ultra-Low Attachment plates in identical media conditions. Growth rate was assessed using cell doubling times and chemoresistance was assessed by determining cell viability in response to a serial dilution of either doxorubicin or cisplatin. Gene expression differences were examined using quantitative reverse-transcription PCR and microarray with principal component and pathway analysis. In-vivo OS xenografts were generated by either subcutaneous or intratibial injection of adherent or AI human OS cells into athymic nude mice. Statistical significance was determined using student’s t-tests with significance set at α = 0.05.ResultsWe show that AI growth results in a global gene expression profile change accompanied by significant chemoresistance (up to 75 fold, p < 0.05). AI cells demonstrate alteration of key mediators of mesenchymal differentiation (β-catenin, Runx2), stemness (Sox2), proliferation (c-myc, Akt), and epigenetic regulation (HDAC class 1). AI cells were equally tumorigenic as their adherent counterparts, but showed a significantly decreased rate of growth in-vitro and in-vivo (p < 0.05). Treatment with the pan-histone deacetylase inhibitor vorinostat and the DNA methyltransferase inhibitor 5-azacytidine mitigated AI growth, while 5-azacytidine sensitized anoikis-resistant cells to doxorubicin (p < 0.05).ConclusionsThese data demonstrate remarkable plasticity in anoikis-resistant human osteosarcoma subpopulations accompanied by a rapid development of chemoresistance and altered growth rates mirroring the early stages of latent metastasis. Targeting epigenetic regulation of this process may be a viable therapeutic strategy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0466-4) contains supplementary material, which is available to authorized users.

This study illustrates that PDX models are suitable surrogates for testing potential therapeutic strategies based on gene expression analysis, modeling clinical drug resistance and hold the potential to assist in guiding prospective patient care.