Owen N. Witte

Tissue recombination models are useful for studying cancer initiation, progression, and metastasis. They also provide an in vivo environment in which to investigate the functional role of stem cells in tissue repair. In this protocol, we describe in detail the dissociated prostate regeneration assay. Dissociated adult murine prostate cells are combined with embryonic urogenital sinus mesenchymal cells and implanted under the renal capsule. Morphological tissue structures with appropriate epithelial–stroma interactions are reconstituted in the grafts.

An appropriate microenvironment provided by the mesenchyme is important for establishing tissue recombination models for epithelial cancer. Urogenital sinus mesenchymal (UGSM) cells derived from embryonic rodent show potent inductive effects for prostate regeneration. Genetic manipulation of these mesenchymal cells allows us to define the contribution of the tumor microenvironment to prostate cancer development. This protocol describes preparation and propagation of murine UGSM cells in culture.

Dominant activating mutations or DNA amplification of tyrosine kinases are not readily observed despite the numerous oncogenic alterations implicated in prostate cancer. We demonstrate that castration resistant prostate cancer (CRPC) exhibits increased tyrosine phosphorylation, raising the question of whether enhanced tyrosine kinase activity is observed in this disease in the absence of specific tyrosine kinase mutations. We generated a mouse model of prostate cancer progression using combinations of commonly perturbed non-tyrosine kinase oncogenes and pathways, including myristoylated Akt (Akt), androgen receptor (AR), Erg, and activated Kras (KrasG12V). We recapitulated the different stages of prostate cancer ranging from prostatic intraepithelial neoplasia (PIN) (Akt) to low grade cancer (Akt+Erg) to highly aggressive prostatic adenocarcinoma (Akt+AR or Akt+KrasG12V). We detected a significant upregulation of tyrosine phosphorylation at the advanced adenocarcinoma stage. Phosphotyrosine peptide enrichment and quantitative mass spectrometry identified oncogene-specific tyrosine kinase signatures, including activation of Egfr Y¹¹⁷² (Akt+Erg), EphA2 Y⁵⁹⁵ (Akt+KrasG12V), and Jak2 Y^1007/08 (Akt+AR). Kinase:substrate relationship analysis of the phosphopeptides also revealed Abl Y²⁴⁵ and Src Y⁴¹⁶ tyrosine kinase activation in Akt+AR tumors; known targets of the tyrosine kinase inhibitor Dasatinib. We are currently evaluating the efficacy of tyrosine kinase inhibitors Dasatinib or Erlotinib on non-tyrosine kinase driven tumors Akt+AR or Akt+Erg, respectively. In summary, the observation of elevated phosphotyrosine signaling in advanced prostate cancer and identification of specific tyrosine kinase pathways from genetically-defined tumor models points to new therapeutic approaches using tyrosine kinase inhibitors for advanced prostate cancer. This abstract is also presented as Poster C35. Citation Format: Justin M. Drake, Jiaoti Huang, Thomas G. Graeber, Owen N. Witte, Nicholas A. Graham, Tanya Stoyanova, Amir Sedghi, Andrew S. Goldstein, Houjian Cai, Daniel A. Smith, Hong Zhang, Evangelia Komisopoulou. Therapeutic targeting of oncogene-specific tyrosine kinase networks during prostate cancer progression [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr PR5.

Cancer cells share multiple characteristics with adult tissue stem cells such as self-renewal, proliferative capacity and differentiation. Several groups have identified Trop2, a type I trans-membrane glycoprotein, as a marker of stem/progenitor cells in adult tissues. Consistent with the relationship between stem cells and tumorigenesis, we have previously identified that Trop2^hi cells are efficient targets for transformation in the prostate. Additionally, Trop2 is highly expressed in various cancers including advanced colorectal, pancreatic, prostate, ovarian and pulmonary carcinomas and is associated with unfavorable outcome. However the functional role of Trop2 in stem cell self-renewal, its relation to transformation, and the molecular mechanisms by which Trop2 transmits signals to regulate these processes remain unclear. We hypothesize that heightened Trop2 may promote the stem-like properties of self-renewal and proliferative activity in prostate cancer. We observe significantly elevated levels of Trop2 in human prostate cancer, lymph node metastases and in mouse models of prostate cancer. Over-expression of Trop2 induces anchorage-independent growth in vitro and murine prostatic intraepithelial neoplasia (mPIN) in vivo. In vitro sphere and in vivo regeneration assays demonstrate that Trop2 regulates stem/progenitor self-renewal in the prostate, suggesting that elevated levels of Trop2 in prostate cancer are selected for by promoting tumor cell self-renewal. Biochemical analysis reveals that Trop2 is activated by regulated intra-membrane proteolysis (RIP), resulting in shedding of the extracellular domain (ECD) and release of the intracellular domain (ICD) to the nucleus. RIP is required for Trop2 activity and cleavage mutants of Trop2 fail to enhance self-renewal in vitro. We generated expression constructs of Trop2 ICD and ECD to test their role in self-renewal and tumorigenesis. Secreted Trop2 ECD induces Trop2 cleavage and results in increased stem/progenitor cell proliferation. Trop2 ICD alone promotes self-renewal activity and transformation as measured by sphere formation and in vivo regeneration assays, respectively. Trop2 ICD is sufficient to induce mPIN lesions similar to previously reported mouse models of prostate cancer. These findings suggest that the ICD is the functional unit of Trop2. Trop2 ICD is involved in a signaling cascade that is dependent upon β-catenin to promote self-renewal. We find Trop2 ICD, but not the ECD, forms a complex with β-catenin. While β-catenin loss does not affect baseline sphere-forming activity, we find that over-expression of Trop2 requires the presence of β-catenin to enhance progenitor activity in vitro. Taken together, our study provides evidence that Trop2 is a key regulator of transformation and self-renewal in the prostate and its function depends on its activation by RIP and downstream signaling through β-catenin. Delineating the molecular mechanism of Trop2 action allows development of therapeutic strategies applicable to not only prostate cancer, but other epithelial malignancies that exhibit increased Trop2 expression. Such strategies include small molecules able to block Trop2 proteolysis, thus inhibiting Trop2 function. More specific therapies such as monoclonal antibodies that will interfere with Trop2 processing and consequent activation could also represent promising future anticancer therapies. Citation Format: Tanya Stoyanova, Andrew Goldstein, Houjian Cai, Justin M. Drake, Jiaoti Huang, Hong Zhang, Owen N. Witte. Trop2 regulates prostate tumorigenesis and stem cell self-renewal [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C70.

Gemcitabine (2′,2′-difluorodeoxycytidine, dFdC) and cytosine arabinoside (cytarabine, ara-C) represent a class of nucleoside analogs used in cancer chemotherapy. Administered as prodrugs, dFdC and ara-C are transported across cell membranes and are converted to cytotoxic derivatives through consecutive phosphorylation steps catalyzed by endogenous nucleoside kinases. Deoxycytidine kinase (DCK) controls the rate-limiting step in the activation cascade of dFdC and ara-C. DCK activity varies significantly among individuals and across different tumor types and is a critical determinant of tumor responses to these prodrugs. Current assays to measure DCK expression and activity require biopsy samples and are prone to sampling errors. Noninvasive methods that can detect DCK activity in tumor lesions throughout the body could circumvent these limitations. Here, we demonstrate an approach to detecting DCK activity in vivo by using positron emission tomography (PET) and ¹⁸F-labeled 1-(2′-deoxy-2′-fluoroarabinofuranosyl) cytosine] ([¹⁸F]FAC), a PET probe recently developed by our group. We show that [¹⁸F]FAC is a DCK substrate with an affinity similar to that of dFdC. In vitro, accumulation of [¹⁸F]FAC in murine and human leukemia cell lines is critically dependent on DCK activity and correlates with dFdC sensitivity. In mice, [¹⁸F]FAC accumulates selectively in DCK-positive vs. DCK-negative tumors, and [¹⁸F]FAC microPET scans can predict responses to dFdC. We suggest that [¹⁸F]FAC PET might be useful for guiding treatment decisions in certain cancers by enabling individualized chemotherapy.

The development of miniaturized cell culture platforms for performing parallel cultures and combinatorial assays is important in cell biology from the single-cell level to the system level. In this paper we developed an integrated microfluidic cell-culture platform, Cell-microChip (Cell-μChip), for parallel analyses of the effects of microenvironmental cues (i.e., culture scaffolds) on different mammalian cells and their cellular responses to external stimuli. As a model study, we demonstrated the ability of culturing and assaying several mammalian cells, such as NIH 3T3 fibroblast, B16 melanoma and HeLa cell lines, in a parallel way. For functional assays, first we tested drug-induced apoptotic responses from different cell lines. As a second functional assay, we performed “on-chip” transfection of a reporter gene encoding an enhanced green fluorescent protein (EGFP) followed by live-cell imaging of transcriptional activation of cyclooxygenase 2 (Cox-2) expression. Collectively, our Cell-μChip approach demonstrated the capability to carry out parallel operations and the potential to further integrate advanced functions and applications in the broader space of combinatorial chemistry and biology.

The epithelium of the adult prostate contains 3 distinct cell types: basal, luminal, and neuroendocrine. Tissue-regenerative activity has been identified predominantly from the basal cells, isolated by expression of CD49f and stem cell antigen-1 (Sca-1). An important question for the field is whether all basal cells have stem cell characteristics. Prostate-specific microarray databases were interrogated to find candidate surface antigens that could subfractionate the basal cell population. Tumor-associated calcium signal transducer 2 (TACSTD2/Trop2/M1S1/GA733-1) was identified because it was enriched after castration, in prostate sphere cells and in the basal fraction. In the murine prostate, Trop2 shows progenitor characteristics such as localization to the region of the gland proximal to the urethra and enrichment for sphere-forming and colony-forming cells. Trop2 subfractionates the basal cells into 2 populations, both of which express characteristic basal cell markers by quantitative PCR. However, only the basal cells expressing high levels of Trop2 were able to efficiently form spheres in vitro. In the human prostate, where Sca-1 is not expressed, sphere-forming progenitor cells were also isolated based on high expression of Trop2 and CD49f. Trop2-expressing murine basal cells could regenerate prostatic tubules in vivo, whereas the remaining basal cells had minimal activity. Evidence was found for basal, luminal, and neuroendocrine cells in prostatic tubules regenerated from Trop2 ^hi basal cells. In summary, functionally distinct populations of cells exist within the prostate basal compartment and an epithelial progenitor can give rise to neuroendocrine cells in vivo.

Adoptive transfer (AT) T-cell therapy provides significant clinical benefits in patients with advanced melanoma. However, approaches to non-invasively visualize the persistence of transferred T cells are lacking. We examined whether positron emission tomography (PET) can monitor the distribution of self-antigen-specific T cells engineered to express an herpes simplex virus 1 thymidine kinase (sr39tk) PET reporter gene. Micro-PET imaging using the sr39tk-specific substrate 9-[4-[(18)F]fluoro-3-(hydroxymethyl)-butyl]guanine ([(18)F]FHBG) enabled the detection of transplanted T cells in secondary lymphoid organs of recipient mice over a 3-week period. Tumor responses could be predicted as early as 3 days following AT when a >25-fold increase of micro-PET signal in the spleen and 2-fold increase in lymph nodes (LNs) were observed in mice receiving combined immunotherapy versus control mice. The lower limit of detection was approximately 7 x 10(5) T cells in the spleen and 1 x 10(4) T cells in LNs. Quantification of transplanted T cells in the tumor was hampered by the sr39tk-independent trapping of [(18)F]FHBG within the tumor architecture. These data support the feasibility of using PET to visualize the expansion, homing and persistence of transferred T cells. PET may have significant clinical utility by providing the means to quantify anti-tumor T cells throughout the body and provide early correlates for treatment efficacy.

We have successfully designed and fabricated an integrated microfluidic platform, the hESC-µChip, which is capable of reproducible and quantitative culture and analysis of individual hESC colonies in a semi-automated fashion. In this device, a serpentine microchannel allows pre-screening of dissociated hESC clusters, and six individually addressable cell culture chambers enable parallel hESC culture, as well as multiparameter analyses in sequence. In order to quantitatively monitor hESC proliferation and pluripotency status in real time, knock-in hESC lines with EGFP driven by the endogenous OCT4 promoter were constructed. On-chip immunoassays of several pluripotency markers were carried out to confirm that the hESC colonies maintained their pluripotency. For the first time, our studies demonstrated well characterized hESC culture and analysis in a microfluidic setting, as well as a proof-of-concept demonstration of parallel/multiparameter/real-time/automated examination of self-renewal and differentiation in the same device.

With their unique ability to differentiate into all cell types, embryonic stem (ES) cells hold great therapeutic promise. To improve the efficiency of embryoid body (EB)-mediated ES cell differentiation, we studied murine EBs on the basis of their size and found that EBs with an intermediate size (diameter 100–300 μm) are the most proliferative, hold the greatest differentiation potential, and have the lowest rate of cell death. In an attempt to promote the formation of this subpopulation, we surveyed several biocompatible substrates with different surface chemical parameters and identified a strong correlation between hydrophobicity and EB development. Using self-assembled monolayers of various lengths of alkanethiolates on gold substrates, we directly tested this correlation and found that surfaces that exhibit increasing hydrophobicity enrich for the intermediate-size EBs. When this approach was applied to the human ES cell system, similar phenomena were observed. Our data demonstrate that hydrophobic surfaces serve as a platform to deliver uniform EB populations and may significantly improve the efficiency of ES cell differentiation.