FIGURES
Figure 1
1A = lack of uniform loss of p-Smad2 in stroma of Tgfbr2-fspko prostate
1B = IHC p-Smad2 of Human benign & PCa prostate
1C = Summary table of % stromal loss of p-Smad2 from TMA sample set
1D = LNCAP/wt stroma, PC3/wt stroma, PC3/NPF p-Smad2 IHC
Figure 2
2A = NPF vs CAF total Smad 1/2/3 IF (cultured in serum)
2B = p-Smad2/Smad2/Actin WB NPF vs CAF
2C = SDF-1 ELISA NPF vs CAF
2D = Wnt3a Elisa NPF vs CAF
Figure 3 = Tissue Recombs
100% WT  WT
100% KO  PIN
50/50 KO/WT  PCa
Figure 4
Figure 5
Figure 6
A
B
Figure 7
A
B (i)
(ii)
(iii)
Figure 8
A
B
Figure 89.
Figure 10
Figure 911
Not done yet with the new model… should be the same though.
Figure 1012
A
Figure 1113
B
C
FIGURE LEGENDS
Figure 1. Loss of TGF-beta signaling in human and mouse prostate tissues. A)
Reduction in phospho-Smad2 staining in stroma associated with adenocarcinoma in
Tgfbr2fspKO prostate. B) IHC demonstrating loss of nuclear p-Smad2 in human PCa but
not benign prostate tissue samples ranked by Gleason score. C) Table summarizing pSmad2 results from human PCa tissue array. D) IHC p-Smad2 in tissue recombs
LNCAP or PC3 w/ WT vs. KO stroma
Figure 2. NPF vs CAF. A) total Smad 1/2/3 IF. B) p-Smad2/Smad2/Actin WB. C)
SDF-1 ELISA. D) Wnt-3a ELISA.
Figure 3.
Percentage of WT and Tgfbr2fspKO stroma in prostate tissue
recombinant sub-renal allografts promotes cancer progression.
A) Normal
glandular architecture observed by H&E of graft of WT prostatic epithelial organoids
recombined with 100% normal Tgfbr2floxE2/floxE2 prostate stromal cells. B) Development
of PIN lesions in grafts of WT prostatic epithelial organoids recombined with 100%
Tgfbr2fspKO prostate stromal cells. C) Progression of adenocarcinoma in grafts of WT
prostatic epithelial organoids recombined with a 50% / 50% mixture of Tgfbr2floxE2/floxE2
and Tgfbr2fspKO prostate stromal cells. Images are representative of sections from N=?
tissue recombinant grafts per experimental group for number? of independent
experiments.
Figure 4. Two-step model of prostate tumorigenesis based on the Tgfbr2fspKO
mouse. Hypothetical mechanism 2 for why SDF production is greatest in mixed stromal cells.
For this case, TGFB knock-out stromal cells produce factor M1 for epithelial tansformation (Step
1) but also products are required from normal stromal cells for epithelial invasion (Step 2).
Figure 5. Establishment of a biologically informed computational model.
Experimental picture (Left) of H&E stained section of mouse prostate with normal
glandular structure comprised of ductal units of basal and luminal epithelial cells
surrounded by basement membrane and well-differentiated smooth muscle (stromal)
cells. Corresponding positions of simulation cells (Right) assuming 50% altered stromal
cells. Epithelial cells are shown in black, normal stromal cells are shown in blue, and
altered stromal cells are shown in cyan.
Figure 6. Results of simulations based upon the established mathematical model
indicate that stromal heterogeneity alters the proliferative and invasive potential
of the prostate epithelial cells. The computational model yields greatest invasion at
heterogeneous mixtures of stromal cells and the extent of epithelial proliferation and invasion
depends on the ratio of the production of morphogens to the threshold response to morphogens.
A) The number of cells that have become proliferative (Step 1, dashed line) and number of cells
that have also become invasive (Step 2, solid line) when the morphogen diffusion lengths of M1
and M2 are 200 and 300 m, respectively and the threshold T required for transformation in
response to M1 and M2 are 0.0453 and 0.3432 morphogen units respectively. The total
abundance of each morphogen produced per source (per cell) is fixed at A=10000 morphogen
units. B) A phase diagram showing the final tissue classification as a function of M1 production by
altered cells (y-axis) and M2 production by normal cells (x-axis) in a tissue that is a 50/50 mix of normal
and altered stroma. If the production rate of both morphogens is low relative to the threshold needed for
transformation, the cells remain normal. Morphogen diffusion lengths and thresholds for M1 and
M2 are as in (A). Error bars indicate the standard error of 100 simulations.
Figure 7. The rate of epithelial cell transformation increases with responsiveness to steady
state morphogen levels. (A) Steady-state morphogen levels of M1 for a random pattern of 50%
mixed altered and normal stromal. The color bar on the right indicates that brighter colored
pixels correspond to higher morphogen concentrations. (B) The state of epithelia cells (normal =
yellow, proliferative = red) when the threshold TM1 required for transformation is (i) 20
morphogen units, (ii) 5 morphogen units or (i) 1 moprhogen units. As the threshold decreases,
epithelia cells transform in response to lower morpphogen levels and the number of transformed
cells increases.
Figure 8. The cell responsiveness to morphogen levels can be tuned to yield experimental
results. In experiments, approximately 80% of epithelial cells became proliferative and 20%
became invasive at a 50-50 mix of altered and normal stroma. A) The threshold of M1 TM1 that
results in 80% transformation of epithelial cells (Step 1) at a 50-50 mix of altered and normal
stroma is shown as a function of the morphogen diffusion length LM1. For a diffusion length of
200 m, the threshold yielding experimental proliferation rates is 0.045 morphogen units (star).
B) The threshold of M2 TM2 that results in 20% transformation of epithelial cells (Step 2) at a 5050 mix of altered and normal stroma when parameters for morphogen M1 are LM1=200 m and
TM1=0.045 morphogen units is shown as a function of the morphogen diffusion length LM2. For a
diffusion length of 300 m, the threshold yielding experimental proliferation rates is 0.343
morphogen units (star). Error bars indicate the standard error of 100 simulations.
Figure 89. Assessment of biological parameters for mRNA and protein
expression of the candidate morphogens, Wnt-3a and SDF-1. Quantitative realtime PCR analysis of mRNA expression for Wnt-3a and SDF-1 from A) unsorted
cultures of Tgfbr2floxE2/floxE2 and Tgfbr2fspKO prostate stromal cells grown separately or as
mixed co-cultures at various ratios in 60mm dishes, B) FACS sorted cells from
unlabeled Tgfbr2floxE2/floxE2 and CMFDA (green) dye labeled Tgfbr2fspKO prostate stromal
cell co-cultures, and C) SDF-1 concentrations measured in conditioned medium from
Tgfbr2floxE2/floxE2 and Tgfbr2fspKO prostate stromal cells grown in culture or co-culture for
72 hr after reaching confluenct by ELISA. Samples were normalized by measurement
of total protein by BCA protein assay.
Figure 10. The cell responsiveness to morphogen levels can not be tuned to yield
experimental results when there is basal production of morphogens. The minimum threshold
that will yield 0% proliferation in the case of 100% normal cells (dotted line) and the maximum
threshold that will yield 80% proliferation in the case of 50% normal cells as a function of the
diffusion length LM1. The abundance of morphogen M1 is AM1=10000 units. There is no
intersection of the graphs, indicating no solution.
Figure 911. Role of tissue position in determining epithelial exposure to
diffusive morphogens and altering transformation and invasive potential.
Averaging results over multiple simulations yields predictions about which epithelia cells are
most vulnerable to transformation. Stromal cells are blue and epithelial cells are black, red or
gray. A) Highest epithelial cell transformation rates in 50/50 mixed population. Red epithelial
cells show cells that transform more than 9 out of 10 simulations. B) Epithelial cell
transformation rates when there is exactly one altered stromal cell. Red epithelial cells show
cells that transform more than 1 out of 10 simulations. Gray epithelial cells show cells that never
transform. Simulation parameters are as in Fig 4A and statistics for coloring are averaged over
100 simulations.
Figure 1012. Model results for different assumptions regarding the production of SDF.
Fraction of epithelia that have become proliferative (dashed line) or invasive (solid line) verses
the fraction of altered stroma if a) altered stromal cells produce SDF, b) altered cells produce
SDF only if there are normal stromal cells, c) altered cells produce SDF in proportion to the
fraction of normal cells. For all cases, morphogen parameters for diffusion length and threshold
L and T are as in Fig 4A. For cases (a) and (b), the abundance of morphogen M2 yielded by each
cell is A =10000 units, and for case (c) the abundance of morphogen M2 is a function of the
fraction of altered stromal where A = 10000*(2-2*fN), where fN is the fraction of normal stroma.
Figure 1113. Projected roles of TGF-B in tumorigenic model.
TABLE LEGENDS