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Cancer Res. Author manuscript; available in PMC 2012 May 15.

Published in final edited form as:

Published online 2011 March 28. doi: 10.1158/0008-5472.CAN-10-2999

PMCID: PMC3096737

NIHMSID: NIHMS284276

Maria A. Kiskowski,^{1,}^{*} Roger S. Jackson, 2nd,^{2,}^{3,}^{*} Jheelam Banerjee,^{2,}^{3} Xiaohong Li,^{2,}^{3} Minchul Kang,^{4} Juan M. Iturregui,^{2,}^{5} Omar E. Franco,^{2,}^{3} Simon W. Hayward,^{2,}^{3} and Neil A. Bhowmick^{6,}^{**}

The publisher's final edited version of this article is available free at Cancer Res

See other articles in PMC that cite the published article.

Prostate cancer develops through a stochastic mechanism whereby pre-cancerous lesions on occasion progress to multifocal adenocarcinoma. Analysis of human benign and cancer prostate tissues revealed heterogeneous loss of TGF-β signaling in the cancer associated stromal fibroblastic cell compartment. To test the hypothesis that prostate cancer progression is dependent on the heterogeneous TGF-β responsive microenvironment, a tissue recombination experiment was designed where the ratio of TGF-β responsive and nonresponsive stromal cells were varied. While 100% TGF-β responsive stromal cells supported benign prostate growth and 100% TGF-β non-responsive stromal cells resulted in pre-cancerous lesions, only the mixture of TGF-β responsive and nonresponsive stromal cells resulted in adenocarcinoma. A computational model was used to resolve a mechanism of tumorigenic progression where proliferation and invasion occur in two independent steps mediated by distinct stromally derived paracrine signals produced by TGF-β non-responsive and responsive stromal cells. Complex spatial relationships of stromal and epithelial cells were incorporated into the model based on experimental data. Informed by incorporation of experimentally derived spatial parameters for complex stromal-epithelial relationships, the computational model indicated ranges for the relative production of paracrine factors by each cell type and provided bounds for the diffusive range of the molecules. Since SDF-1 satisfied model predictions for an invasion promoting paracrine factor, a more focused computational model was subsequently used to investigate if SDF-1 was the invasion signal. Simulations replicating SDF-1 expression data revealed the requirement for cooperative SDF-1 expression, a prediction supported biologically by heterotypic stromal IL-1β signaling between fibroblastic cell populations. The cancer stromal field effect supports a functional role for the unaltered fibroblasts as a cooperative mediator of cancer progression.

Organ development and epithelial tumor progression are the result of reciprocal and sequential stromal-epithelial interactions. Prostate cancer (PCa), the most common non-cutaneous cancer in men, is initiated by the transformation of luminal epithelial cells that line the prostatic ducts resulting in altered proliferation leading to prostatic intra-epithelial neoplasia (PIN) (2). Loss of cell polarity and invasion through the basement membrane into the surrounding prostatic stroma by the transformed epithelia mediates the establishment of prostatic adenocarcinoma. The sequential progression of epithelial differentiation to adenocarcinoma is dictated by its inherent genetic stability as well as the cues provided by the surrounding microenvironment (3–5). Tgfbr2^{fspKO} mice, with a conditional knockout (KO) of the transforming growth factor beta (TGF-β) type II receptor (TβRII) gene (*Tgfbr2*) in stromal fibroblasts, develop PIN lesions and subsequently prostate adenocarcinoma (6, 7). Human PCa is analogously associated with the heterogeneous loss of TβRII expression in the stromal compartment (7, 8).

We used a flexible, biologically intuitive, hybrid computational model to investigate mechanisms for cell-cell communication in stromal and epithelial compartments on tumor initiation and progression. Hybrid mathematical modeling has developed into a means of explaining biologic mechanisms, combining explicit cell representations with continuum-based biological descriptions (11, 14–16). Our hybrid computational model combines a discrete model for the differentiation of individual epithelial cells with a continuum model of stromally derived signaling factor diffusion. Secreted signaling factors in this study include paracrine factors (that traverse the basement membrane between the stroma and the epithelia) and heterotypic stromal signaling factors (that communicate between populations of fibroblastic cells). Herein we report that biological experiments (i*n vivo* tissue recombination and *in vitro* biological models) and computational mathematical modeling together test and support hypotheses for inter-cellular signaling within a heterogeneous prostatic stromal compartment and prostatic epithelial progression to adenocarcinoma.

Our computational model couples a discrete, stochastic description of cells at the macroscopic scale with a continuous, deterministic description of paracrine factor diffusion at the microscopic scale.

A spatially explicit model for cell positions incorporates the spatial scales of epithelial and stromal cells located at discrete lattice locations based on their positions in a histologic cross-section of mouse prostate (epithelia circumscribing prostate ducts surrounded by stromal fibroblastic cells. Fibroblasts were randomly identified as dominant point sources for either *M*_{1} or *M _{2}* paracrine factors, with probabilities depending upon the simulated fraction of WT and Tgfbr2-KO fibroblasts, respectively.

Forty-four stromal cells were point sources for paracrine factors *M*_{1} and *M*_{2}. We denote the set of wild-type (normal (N)) and Tgfbr2-KO (altered (A)) stromal cell positions as *S _{N}* and

To model diffusion in the context of the complex geometry of the mouse prostate, paracrine factors diffuse in the region between and surrounding (but not within) irregularly shaped ducts. In normal prostate, the basement membrane provides a diffusion barrier between epithelial and stromal cells. The effect of the basement membrane as a barrier to diffusion and the erosion of the basement membrane were not modeled here. Instead, the model considers the steady-state levels of paracrine factors and their effect on epithelial cells with the assumption that any erosion of the basement membrane had already occurred. Since *M*_{1} and *M*_{2} diffuse freely in the inter-ductal space, with certain metabolic decay rates, the kinetics of *m*_{1} and *m*_{2} can be described as uncoupled reaction diffusion:

$$\begin{array}{l}\frac{\partial {m}_{1}}{\partial t}={k}_{1}{\displaystyle \sum _{i=1}^{n}\delta \left({x}_{i},{y}_{i}\right)}-{k}_{d1}{m}_{1}+{D}_{1}{\nabla}^{2}{m}_{1}\hfill \\ \frac{\partial {m}_{2}}{\partial t}={k}_{2}{\displaystyle \sum _{i=1}^{a}\delta \left({\overline{x}}_{i},{\overline{y}}_{i}\right)}-{k}_{d2}{m}_{2}+{D}_{2}{\nabla}^{2}{m}_{2}\hfill \end{array}$$

Equation Set 1:

Here *k _{1}* and

- $\frac{\partial {m}_{1}}{\partial n}=0$
- $\frac{\partial {m}_{2}}{\partial n}=0$, where
*n*indicates the outward facing normal of the boundary

Epithelial cells respond to paracrine factors by transforming from normal to proliferative at threshold levels of *M*_{1} and from proliferative to invasive at threshold levels of *M*_{2}. Levels of paracrine factors are measured at steady state concentrations. This simplification is justified if the time scales of epithelial transformation are much slower than the time scales of paracrine factor diffusion, which is reasonable as diffusive factor steady state concentrations establish at fast time scales compared to cellular responses (17, 18). Normal (*N*), proliferative (*P*) and invasive (*I*) cells are labeled as *E _{N}*,

$$\begin{array}{l}{D}_{1}{\nabla}^{2}{m}_{1}^{\infty}={k}_{{d}_{1}}{m}_{1}^{\infty}-{k}_{1}{\displaystyle \sum _{i=1}^{a}{\delta}_{\left({x}_{i},{y}_{i}\right)}}\hfill \\ {D}_{2}{\nabla}^{2}{m}_{2}^{\infty}={k}_{{d}_{2}}{m}_{2}^{\infty}-{k}_{2}{\displaystyle \sum _{i=1}^{n}{\delta}_{\left({\overline{x}}_{i},{\overline{y}}_{i}\right)}}\hfill \end{array}$$

Equation Set 2:

Next, the proposed underlying mechanism for epithelial transformation can be written:

$$\begin{array}{l}{E}_{N}+{M}_{1}^{*}\to {E}_{P}\hfill \\ {E}_{P}+{M}_{2}^{*}\to {E}_{I}\hfill \end{array}$$

Equation Set 3:

for quasi-steady paracrine factor levels ${M}_{1}^{*}$ and ${M}_{2}^{*}$ that were above threshold levels *T _{M1}* and

$${E}_{N}\stackrel{M{*}_{1}}{\to}{E}_{P}\stackrel{M{*}_{2}}{\to}{E}_{I}$$

(3b)

On the other hand, for ${M}_{1}^{\circ}$ and ${M}_{2}^{\circ}$ below threshold levels:

$$\begin{array}{l}{E}_{N}+{M}_{1}^{\mathrm{o}}\to {E}_{N}\hfill \\ {E}_{P}+{M}_{2}^{\circ}\to {E}_{P}\hfill \end{array}$$

(3c)

Assuming *E _{N}*,

$$\begin{array}{l}\frac{d{e}_{n}}{dt}=-{k}_{N}\xb7H({m}_{1}^{\infty}-{m}_{1}^{*})\xb7{m}_{1}^{\infty}\xb7{e}_{n}\hfill \\ \frac{d{e}_{p}}{dt}=-{k}_{P}\xb7H({m}_{2}^{\infty}-{m}_{2}^{*})\xb7{m}_{2}^{\infty}\xb7{e}_{p}+{k}_{N}\xb7H({m}_{1}^{\infty}-{m}_{1}^{*})\xb7{m}_{1}^{\infty}\xb7{e}_{n}\hfill \\ \frac{d{e}_{i}}{dt}={k}_{P}\xb7H({m}_{2}^{\infty}-{m}_{2}^{*})\xb7{m}_{2}^{\infty}\xb7{e}_{p}\hfill \end{array}$$

Equation Set 4:

where *k _{N}* and

Immunofluoresence (for CD90 and phosphorylated-Smad2) and immunohistochemistry (for phosphorylated-Smad2) were performed on paraffin-embedded mouse or human prostate tissue sections as described (7, 19). A pathologist blindly evaluated immunohistochemical staining. Primary stromal cells were isolated, cultured, and recombined with prostatic epithelial organoids as described (7, 19, 20). Tissue recombinations were performed as previously described (19, 21). For inhibition of DNA methylation, stromal fibroblasts were cultured for five days in the presence of 10 µM 5-aza-deoxycytidine (Sigma). Tgfbr2^{fspKO} prostate stromal cells labeled with 2.0 µM CMFDA dye (Molecular Probes, Invitrogen) were co-cultured with Tgfbr2-flox stromal cells in 100 mm tissue culture dishes at 2×10^{6} cells per dish for 20hrs. Cells were sorted on a BD FACS Aria II into cold RNA extraction buffer. DNase-I treated total RNA was isolated using the Qiagen RNeasy kit as instructed. Expression of SDF1, SFRP1, SFRP2, β-Actin, and GAPDH mRNA were evaluated by Quantitative Real-Time PCR (qRT-PCR). Amplification specificity was confirmed by melt-curve analysis and agarose gel visualization. Expression values were normalized for relative expression using the ΔΔC_{T} method. PCR primer sequences, numerical scheme, and steady state analysis methods are found in supplemental information. All procedures for human tissue acquisition and animal procedures were approved by the Vanderbilt University Institutional Review Board and Vanderbilt Institutional Animal Care and Use Committee, respectively.

To extend our study of stromal TGF-β signaling, phosphorylated-Smad2 expression was tested in a tissue microarray consisting of 80 human PCa samples. Independent of Gleason grade, phosphorylated-Smad2 was expressed homogenously throughout the epithelial compartment of all prostate samples examined. However, the stromal fibroblastic cells had heterogeneous phosphorylated-Smad2 staining in PCa tissues (Fig. 1A). Corresponding tissues of benign prostate hyperplasia (BPH) had a more homogeneous phosphorylated-Smad2 expression among stromal fibroblasts. Previous studies have suggested CD90 to be a marker heterogeneously expressed in PCa associated stromal cells (22). To further explore the role of stromal TGF-β signaling in PCa progression, we co-localized CD90 and phosphorylated-Smad2 expression by immunofluorescence in human PCa associated (N=10 patient samples, Gleason score range: 7–9) and non-cancer associated (N=6 patient samples) prostatic stromal fibroblastic cells. Interestingly, CD90 and phosphorylated-Smad2 expression was heterogeneous in the stromal cells of human PCa tissues (Fig. 1B). CD90 expression was generally limited to cancer associated stromal cells lacking phosphorylated-Smad2 expression. Stromal compartments associated with benign epithelia expressed both CD90 and phosphorylated-Smad2.

Tgfbr2^{fspKO} mice were also found to have a heterogeneous loss of downstream Smad2 activation in stromal fibroblasts (Fig. 1C), partially attributable to the FSP1 promoter being active in only 40–60% of prostate fibroblasts. To determine the biological ramification of the observed stromal heterogeneity in TGF-β signaling, tissue recombination allografting experiments were conducted (N=6 grafts per group). Prostatic stromal cells from control Tgfbr2^{floxE2/floxE2} mice (Tgfbr2-flox), and those from Tgfbr2^{fspKO} (Tgfbr2-KO) were cultured. Although the loss of stromal heterogeneity is not observed *in vivo*, in the context of stromal-epithelial interactions, the culturing of prostatic stromal cells from Tgfbr2^{fspKO} mice beyond six passages results in *Tgfbr2* knockout in 100% of the cells (19). The recombination of WT prostatic epithelial organoids with Tgfbr2-flox stroma under the renal capsule of syngeneic mice for 12 weeks yielded morphologically normal prostatic tissues (Fig. 2). In contrast, recombination of WT prostatic epithelial organoids with Tgfbr2-KO stromal cells resulted in PIN development in the same time period. The prostatic recombinants resulting from the equal mixture of Tgfbr2-flox and Tgfbr2-KO stromal cells with WT prostatic epithelia resulted in prostatic adenocarcinoma. The most immediate interpretation of this striking observation is that the epithelia transform from normal to aggressive in two steps, where in the first step Tgfbr2-KO stromal cells contribute to initiating epithelial transformation and in the second step Tgfbr2-flox stromal cells contribute to the progression of initiated epithelia to an aggressive phenotype (invasion). Further, we believe it is likely that stromal-epithelial interactions are mediated by diffusive factors of some kind (23). Thus, we hypothesized a diffusive, two-step mechanism for epithelial transformation dependent upon stromal heterogeneity (TGF-β responsive and nonresponsive stromal cells). In step 1 of the hypothesized model, TGF-β non-responsive (Tgfbr2-KO) stromal cells produce a diffusive paracrine factor-1 (*M _{1}*), which cause epithelial cells to transform from normal to proliferative. In step 2, TGF-β responsive stromal cells (wild type and Tgfbr2-flox) produce a diffusive paracrine factor-2 (

A mathematical model describing epithelial and stromal fibroblastic cell signaling interactions was developed to test the viability of this proposed mechanism for epithelial transformation. Stromal and epithelial cell positions were modeled according to a histological section of mouse prostate (Fig. 3A) and simulations were used to investigate the predictions of a model for paracrine interaction. The model assumed epithelial cells transformed in response to threshold levels of stromal factors at steady-state concentrations (see the Quick Guide to Equations and Assumptions).

Implementation of the two-step mechanism in the computational model yielded results that qualitatively reproduced experimental observations over a wide range of simulation parameters. In simulations, the ratio of WT and Tgfbr2-KO stromal cells was varied from 0 to 1. The measured response variable for all simulations was the fraction of epithelial cells that transformed (Step 1, proliferative or Step 2, invasive). The fraction of transformed epithelial cells for a typical set of simulation parameters is shown in Fig. 3B. For 100% WT stromal cells (fraction altered stroma, FAS=0.00), there were no associated proliferative (Step 1) or invasive (Step 2) epithelial cells. For 100% Tgfbr2-KO stromal cells (FAS=1.00), there were only normal and proliferative epithelia. The computational model yielded invasion only at heterogeneous mixtures of stromal cells. Key model parameters (paracrine factor diffusion length ** L**, paracrine factor abundance

Parameters for the computational model were chosen to match experimental results quantitatively, but parameters were not constrained uniquely. It was experimentally observed that at a 1:1 WT : Tgfbr2-KO stromal fibroblastic cell ratio, the majority of epithelial cells became proliferative while a minority of epithelial cells became invasive. Model parameters could be tuned to match experimental epithelial transformation rates. Since increasing the diffusion length, increasing the abundance, and decreasing the threshold level all have the effect of increasing the fraction of cells that transform; this tuning was non-unique. In the Supplemental Information (SI), we describe how we tuned the parameters to find a non-unique set that generated and matched experimental results for the fraction of proliferative and invasive epithelial cells (SI Fig. 1, SI Fig. 2), and that the model predicted tissue morphology and stromal cell density influence upon epithelial progression toward cancer (SI Fig. 3).

In our proof-of-principle consideration of the two-step model for tumorigenesis, we implemented the assumption that the paracrine factor produced by Tgfbr2-KO fibroblasts that supported proliferation (*M _{1}*) was not produced by WT fibroblasts at any level, and likewise that the paracrine factor produced by WT fibroblasts that supported invasion (

Incorporation of experimental data (for the extent of epithelial transformation and the spatial scale of stromal-epithelial interaction) into the model established upper and lower boundaries for the threshold for epithelial transformation (see SI Fig. 4 and SI Table 1 for a more detailed explanation) and thereby the model was able to establish limits upon the relative n-fold increase in paracrine factor production and diffusion length required to obtain either no transformation (*Outcome A*) or epithelial transformation (*Outcome B*). Given a known fraction of epithelial transformation in *Outcome B*, the model predicted a lower bound for the fold difference in paracrine factor production between *Outcomes A* and *B*. If the fold difference in paracrine factor production was also known, the model made a more detailed prediction about the diffusion length of the invasive factor (*M _{2}*). Since the relative production of factors by each cell type may be measured experimentally, the permitted ranges predicted by the model may be used to evaluate whether putative factors for proliferation or invasion would be consistent with the diffusive two-step mechanism for tumorigenesis [as we describe below for Wnt-3a (

Wnt-3a is a secreted factor dominantly expressed by stromal cells that is a candidate for supporting proliferation (7, 19, 24). Further, we identified that Tgfbr2-KO stromal cells up regulated Wnt signaling by the silencing of its antagonists, SFRP1 and SFRP2. Interestingly, the treatment of Tgfbr2-KO stromal cells with the DNA methylation inhibitor, 5-aza-deoxycytidine (5-aza-dC), restored SFRP1 and SFRP2 mRNA expression nearly to that observed for WT stromal cells (Fig. 4). Similarly, Tgfbr2-KO stromal cells epigenetically down-regulate SDF-1 expression, also restored by 5-aza-dC treatment. Thus, Tgfbr2-KO stroma supported paracrine Wnt signaling, whereas, the WT stroma promoted SDF-1 expression.

In our tissue recombination allografting experiments, it appeared that more than 80±15% of epithelial cells transformed to become proliferative in the case of organoids with Tgfbr2-KO stromal cells. Our computational model thus predicted that the production of the factor in tissues supporting proliferation must be more than 12-fold that of tissues not supporting proliferation. However, we previously found that the production of Wnt-3a by WT stromal cells was 20% that of Tgfbr2-KO stromal cells (7) resulting in only a 3-fold difference in factor production in the tissues supporting and not supporting proliferation (Table 1). In other words, model parameters could not be found for *M _{1}* (diffusion rate, decay rate, and thresholds for transformation) such that 80% of the epithelial cells would become proliferative at the 50/50 mixture of stromal cell types but 0% would become proliferative when the stromal cells are 100% normal. Indeed, we found that over a large parameter range (e.g., diffusion lengths ≤ 5000 µm), the model could not accommodate this level of

The chemokine, SDF-1/CXCL12 is implicated as a promoter of tumor epithelial progression to an invasive phenotype (25, 26). Expression of SDF-1 and its receptor (CXCR4) is positively regulated by TGF-β signaling (27, 28). Previously, we have performed microarray analysis of laser-capture microdissected (LCM) Tgfbr2^{floxE2/floxE2} and Tgfbr2^{fspKO} prostate stromal cells (data available in GEO database record GSE22130) isolated from mice (29). The data revealed the heterogeneity of gene expression of the stroma as well as a 77-fold increase in SDF-1 expression by Tgfbr2^{fspKO} prostate stroma. This significant induction of SDF-1 mRNA expression in a heterogeneous stromal microenvironment is consistent with SDF-1 as a candidate paracrine factor in the promotion of PCa progression. Independently, SDF-1 mRNA levels in isolated cultures of WT and Tgfbr2-KO prostate stromal cells were quantified by real-time PCR. WT prostate stromal cells expressed 4-fold higher levels of SDF-1 mRNA than Tgfbr2-KO prostate stromal cells when grown alone in culture. The differential expression of SDF-1 here as compared to that measured by the microarray may be due to the presence of additional *in vivo* stromal cell types isolated by the LCM technique as compared to an isolated pure fibroblastic population grown in culture.

In our tissue recombination allografting experiments, it appeared that more than 20±5% of epithelial cells transformed to become invasive in the case of organoids with an equal mix of both stromal cells. Our computational model predicted that the production of the paracrine factor supporting invasion by the WT cells must be more than 2-fold that produced by the Tgfbr2-KO stroma. Thus, SDF-1 was consistent with *M _{2}* in the two-step mechanism for tumorigenesis since the production of SDF-1 by Tgfbr2-KO cells is only 25% that of the WT cells. In other words, model parameters can be found for

To quantify SDF-1 expression by the individual stromal fibroblastic cell, co-cultured mixtures of fibroblasts were separated by FACS sorting based on CMFDA (green) dye labeling of the Tgfbr2-KO cells, and SDF-1 mRNA quantified by qRT-PCR. The co-cultures were designed at the previously described ratios of Tgfbr2-KO and WT stromal cells (see Fig. 3B). FACS sorting of WT stromal cells grown in 75%:25% and 50%:50% co-culture with Tgfbr2-KO stromal cells revealed an approximately 5-fold and 8-fold increase in SDF-1 mRNA expression, respectively, compared to Tgfbr2-KO cells grown alone (Fig. 5C). Interestingly, SDF-1 mRNA levels of the Tgfbr2-KO stromal cells were elevated approximately 2-fold as a result of co-culture over Tgfbr2-KO cells grown alone. The results suggested that both WT and Tgfbr2-KO stromal cells may cooperatively induce production of one or more heterotypic stromal signaling factors resulting in increased SDF-1 mRNA expression. Inputting the experimentally measured relative production rates of SDF-1 for paracrine factor *M _{2}* production levels in the computational model resulted in the experimentally observed rate of epithelial transformation (SI Fig. 5).

SDF-1 up-regulation by WT stroma in the case of a mixed stromal cell population compared with 100% WT stroma indicated that the presence of Tgfbr2-KO cells is communicated in some way to the WT cells, suggesting, for example, that there is an heterotypic stromal signaling factor (*M _{3}*) secreted by the Tgfbr2-KO cells that WT stromal cells respond to by up-regulating SDF-1 expression. Simulations tested the hypothesis that an

The potential for a Tgfbr2-KO-derived *M _{3}* was tested in WT stromal cells. Microarray analysis of laser-capture microdissected prostate stroma from Tgfbr2-WT and Tgfbr2-KO mice revealed altered production of multiple secreted factors including the cytokines IL-1α (6-fold reduction), IL-β (4-fold reduction), and IL-6 (53-fold induction) by Tgfbr2-KO stroma in comparison to the Tgfbr2-WT stroma. Each of these factors have been implicated in the regulation of SDF-1 in the literature (30–32). Together these data imply that IL-1α, IL-1β, and Il-6 could be candidate heterotypicstromal signaling factors regulating SDF-1 expression.

We describe a biological model for tumor initiation and progression based on heterogeneous stromal production of diffusive factors that mediate epithelial transformation. The aim was to account for a striking set of observations in which heterogeneity (but not homogeneity) of stromal fibroblastic cells was associated with tumorigenesis. Given obtained biological data for rates of epithelial transformation, the model predicted ranges for the relative fold production of factors. Given experimental measures of factor production, the model reported whether the factor was consistent with the biological model and predicted its diffusive range.

Given the ternary observations of the tissue recombination allografting experiments, it seemed unlikely that alternative hypotheses to the proposed mechanism (such as the epithelial progression does not occur in at least two steps or that these steps are not mediated by the stroma) would hold true. Certainly, the mechanism may be more complex, with added layers of interaction. Much literature supports that alterations in the stroma alone, independent of genetic events in the epithelium, can promote progression of neighboring epithelium to cancer (6, 21, 33–37). The step-wise nature of human carcinoma progression has been highlighted by the Knudson “Two Hit Hypothesis” (38). Paracrine-mediated cancer initiation and progression likely involves at least two distinct steps perpetuated by two or more factors produced by at least two independent cell types. Fibroblasts represent a heterogeneous cell population, some of which express FSP-1 and accordingly were knocked out for Tgfbr2 expression in the Tgfbr2^{fspKO} mice. At least two fibroblastic cell sub-populations could be distinguished by CD90 expression (17) and we found CD90 expression coincided with heterogeneous Smad2 activity loss in human PCa associated fibroblasts. Similar fibroblastic heterogeneity in TGF-β responsiveness occurs in Tgfbr2^{fspKO} mouse prostates and is likely functionally essential for the ensuing cancer progression.

The model is valid to the extent that stromal interactions were dominated by diffusive signaling. This biologically informed computational model was independent of specific paracrine factor production. *M _{1}* or

With respect to details of factor production and number, the computational model is flexible and may be modified to accommodate any set of experimental observations regarding factor production for a report of the viability of the model with these factors. Here, the model was modified to accommodate non-linear, cooperative levels of *M _{2}* production as observed in SDF-1 expression. Inputting the production levels phenomenologically, showed that SDF-1 was consistent with the model

The dichotomous view of TGF-β action as a tumor-suppressor in benign tissues and tumor-promoting in initiated cancer can involve the fibroblasts. TGF-β is tumor-suppressive in WT fibroblasts since it represses the secretion of Wnt-3a (and other Stat3 induced paracrine factors) (6, 7, 18, 32). TGF-β promotes SDF-1 production by fibroblasts and the expression of its receptor, CXCR4, in epithelial cells (33). The TGF-β non-responsive fibroblasts associated with cancerous epithelia express factors like IL-1β (*M _{3}*) to further SDF-1 production. Such TGF-β responsive heterotypic fibroblastic and stromal-epithelial signaling would support PCa onset and progression (Fig. 6B).

Previous studies have emphasized the critical role of carcinoma-associated fibroblasts in cancer progression. The data suggest that the unaltered WT fibroblasts in the heterogeneous mixture of fibroblastic cells that make up the cancer stroma are not merely witnesses, but contribute to pro-carcinogenic cues that initiate epithelia, establishing a stromal field effect (39). Cooperativity of signaling within a heterogeneous stromal compartment would provide a mechanism whereby one could explain the multi-focal and polyclonal progression of PCa (39, 40). This can be envisioned where an initiating event in a particular duct confined prostatic epithelial cell causes a change in the neighboring stroma and in turn potentiates initiating change(s) in the adjacent epithelia at a somewhat distant ductal site(s).

A computational model was used to resolve a mechanism of prostate epithelial tumor initiation dictated by paracrine signaling. Model simulations and biological experiments supported fibroblast responsiveness to TGF-β as an initial tumor suppressor and subsequent mediator of tumor progression. TGF-β non-responsive stromal cells permitted the initial proliferative step. Cooperativity of both TGF-β non-responsive and responsive stromal cells was required for the secondary invasion step. Thus, stromal heterogeneity in TGF-β responsiveness enables a paracrine mediated tumorigenesis.

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The authors wish to thank the members and advisors of the Vanderbilt University Tumor Microenvironment Network (VUTMEN) for helpful discussions throughout the course of this work. We thank Anne Kenworthy (Vanderbilt University) for being supportive of Minchul Kang’s assistance with this work. This work was supported by NIH/NCI RO1-CA108646 and U54-CA126505 (to N.A.B.) and a DOD-PRCP postdoctoral training grant W81XWH-08-1-0542 (to R.S.J.).

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