Figure 1.
(A) Evaluation of p75 receptor expression in p75 shRNA transfected tumor cells by western blot analysis. Lanes 1, 2, 3 and 4 were transfected with various combinations of p75 target shRNA as previously described [13]. To confirm specificity of the bands we ran positive and negative control protein lysates recommended by the p75 antibody manufacturer. Actin expression was used as loading control. Compared to the expression of p75 receptor in intact tumor cells (lane 5) target sequences of p75 shRNA #1 and #3 showed no detectable expression of TNF receptor p75 (compare lane 5 to lanes 1 and 3). (B) Experimental design and body weight change over the course of the study. Intact LLC (LLC) and LLC with knockdown of TNFR2/p75 (p75KD/LLC) were inoculated into mice flanks (1 × 106 cells). LLC group consisted of WT mice that were injected with intact LLCs (n = 15) and p75KD/LLC consisted of WT mice that were injected with stably transfected (≥90%) p75KD/LLCs (n = 15). The two major groups WT host/LLC and WT host/p75KD/LLC were further divided into four groups: LLC minus (−) TNF consisted of WT mice inoculated with intact LLC that were injected with saline (n = 5), LLC plus (+) TNF consisted of WT mice with intact LLC that were injected with rmTNF (n = 10), p75KD/LLC-TNF consisted of WT mice with p75KD/LLC that were injected with saline (n = 5) and p75KD/LLC+TNF consisted of WT mice with p75KD/LLCs that were injected with rmTNF (n = 10). Tumor growth was monitored on a daily basis post-inoculation. Body weight data were plotted as a graph between tumor volume (mm3) and time period after tumor inoculation for all groups. Tumors, including peri-tumoral stroma, were carefully bisected to make sure that tumor structure is intact and tumors were weighted. Tumors, femurs, spleens and peripheral blood were collected for histology staining to evaluate possible treatment toxicity and inflammatory responses.
Figure 2.
Flank tumor appearance at the end of the study and graphic representation of tumor volumes and weights. (A)
Representative images of mice with flank tumors in (left to right) intact LLC in WT host injected with saline; p75KD/LLC in WT host injected with saline showing that knocking down p75/TNFR2 in LLC does not affect LLC growth in the WT host; intact LLC in WT host injected with rmTNF showing that injecting low dose exogenous rmTNF stimulates WT LLC growth in WT host; and p75KD/LLC in WT host injected with rmTNF showing that knocking down p75/TNFR2 in LLC and injecting very low dose of exogenous rmTNF significantly inhibits LLC growth in WT host. (B) Flank tumor volumes collected from 5–10 mice/treatment group before the first rmTNF injection (day 8 after initial tumor inoculations) and at end of the study (day 15 after initial inoculations). (C) Graphic representation of completely bisected flank tumor weights data collected from 5–10 mice/treatment at end of the study.
Figure 3.
Representative images of tumor H&E stained sections from four treatment groups, light microscopy at ×40 magnification. (A) Intact LLC in WT host injected with saline - viable tumor composed of highly pleomorphic malignant epithelial cells and brisk mitotic index. No necrosis seen. (B) p75KD/LLC in WT host injected with saline - viable carcinoma showing high mitotic activity and rare apoptotic bodies. (C) Intact LLC in WT host injected with rmTNF - partially viable carcinoma with focal necrosis and mild inflammatory change. (D) p75KD/LLC in WT host injected with rmTNF - massively necrotic tumor with no viable cells present. There was moderate acute inflammatory infiltrate in the tumor tissue. Please note that dotted circles in A, B and C indicate representative mitotic tumor cells.
Table 1.
Summary of tumor tissue morphologic assessment.
Figure 4.
Evaluation of tumor and EC apoptosis.
Apoptosis and tumor angiogenesis was evaluated in tumor tissues by triple immunostaining with terminal transferase dUTP nick end labeling (TUNEL), CD31 and Topro-3. The tumor area was identified by H&E staining of adjacent sections. (A–D) Representative images of triple-immunostained tumors for TUNEL (red), CD31 (green) and Topro-3 (blue); Insets identified by dashed squares in A–D indicate higher magnification of the selected areas in solid squares. Arrowheads indicate TUNEL (+) cells (red); block arrows indicate CD31 (+) cells (green) and arrows indicate double TUNEL/CD31 (+) cells (red/green and yellow). (E) Quantification and graphic representation of only TUNEL (+) cells in all four treatment groups. (F) Quantification and graphic representation of double TUNEL/CD31 (+) cells in all four groups.
Figure 5.
Numerical simulation results of the mathematical model.
Total tumor populations (black solid curves) made up of viable tumor cells (V, green) and necrotic cells (N, red) quickly approach vascular carrying capacity (K, grey) and continue to growth after carrying capacity increase through host angiogenic response to necrosis-secreted TNF (F, grey dot-dashed). (A) Slowly emerging necrotic cells secrete TNF (F, grey dot-dashed) that stimulates transient angiogenesis through host cells and p75-competent cancer cells. Intact LLC tumor volume closely follows the increasing carrying capacity. Final necrotic tumor fraction is ∼2%. Experimentally measured tumor volumes (grey box plots) shown for model validation. (B) p75KD-LLC tumor growth dynamic mimic intact LLC growth. Smaller tumor growth due to impaired pro-angiogenic signaling through p75. Final necrotic tumor fraction is ∼7%. Experimentally measured tumor volumes (blue box plots) shown for model validation. (C) Carrying capacity transiently increases through injection of rmTNF (F, grey dot-dashed, in blue highlighted time interval) initially stimulating tumor growth. Increase in necrotic mass limits tumor growth to below carrying capacity. Final necrotic tumor fraction is ∼19%. Experimentally measured tumor volumes (red box plots) shown for model validation. (D) Carrying capacity transiently increases through injection of rmTNF (F, grey dot-dashed, in blue highlighted time interval) initially stimulating p75KD/LLC+rmTNF tumor growth and later dwarfing tumor growth through TNF-induced cell death and increasing necrosis. Final necrotic tumor fraction is ∼39%. Experimentally measured tumor volumes (magenta box plots) shown for model validation. Model parameters: α = 10, β = 0.06, γ = 0.02, ζ = 0.5, δ = 6.2, ηh = 6, ηc = 0.025 (ηc = 0 for p75KD/LLC+rmTNF), θ = 0.24, ε = 6 (ε = 0 on non-treatment days), ω = 0.003, φ = 0.02.
Figure 6.
Evaluation of possible exogenous rmTNF toxicity in bone marrow and spleen.
Representative images of H&E stained bone marrow and spleen tissue. (A–B) Bone marrow - There was granulocytic hyperplasia in the BM of mice with necrotic tumors in rmTNF-injected groups, reflected by a shift of the myeloid/erythroid ratio (∼3:1 vs. ∼8:1) in the BM (erythroid islands indicated within the encircled areas). (C–F) Spleen - There was a marked increase in extramedullary hematopoiesis in spleens of mice with necrotic tumors in rmTNF injected indicated by the cellularity within the encircled regions between normal lymphoid tissue (white pulp).