Trp53 inactivation in the tumor microenvironment promotes tumor progression by expanding the immunosuppressive lymphoid-like stromal network

Abstract

Inactivation of the tumor suppressor p53 through somatic mutations, observed in 50% of human cancers, is one of the leading causes of tumorigenesis. Clinical and experimental evidence also reveals that p53 mutations sometimes occur in tumor-associated fibroblasts, which correlate with an increased rate of metastases and poor prognosis, suggesting that p53 dysfunction in the tumor microenvironment (TME) favors tumor establishment and progression. To understand the impact of p53 inactivation in the TME in tumor progression, we compared the growth of subcutaneously inoculated B16F1 melanoma in p53(null) and wild-type (WT) mice. Interestingly, tumor growth in p53(null) mice was greatly accelerated, correlating with marked increases in CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSC), FoxP3(+) regulatory T cells, and a loss of effector function, compared with those in WT mice. This augmented immunotolerant TME in p53(null) mice was associated with a marked expansion of a specialized stromal network in the tumor and spleen. These stromal cells expressed markers of fibroblastic reticular cells of lymphoid organs and were readily expanded in culture from p53(null), but not WT, mice. They produced high levels of inflammatory cytokines/chemokines and immunosuppressive molecules, thereby enhancing MDSC differentiation. Furthermore, they significantly accelerated tumor progression in WT mice when co-injected with B16F1. Together, our results show that tumor-stroma interaction in hosts with dysfunctional p53 exacerbates immunosuppression by expanding the lymphoid-like stromal network that enhances MDSC differentiation and tumor progression.

Fig. 1. Trp53^null hosts augment tolerogenic tumor microenvironment and promote tumor progression

WT and p53^null C57/B6 mice were inoculated subcutaneously with 2 × 10⁵ B16F1 cells. (A) Tumor size was measured every other day (n=10 - 15). (B) Mice were euthanized on day18 post-inoculation. CD4⁺, CD8⁺, CD49b⁺, and CD11b⁺ cells among tumor infiltrating leukocytes (TIL, CD45⁺) were examined. (C) The percentage of effector cytokine producing TIL-CD4 and (D) FoxP3⁺ regulatory T cells was determined. Results are presented as mean ± standard error (s.e.) of 5 – 10 mice from at least three independent experiments. * represents a significant difference (p < 0.05). Two-tailed Student’s t-test.

Fig. 2. The accelerated tumor progression in p53^null hosts is associated with an increase in MDSCs and expansion of lymphoid-like stromal network

(A) TIL-MDSCs and subpopulations from WT and p53^null mice were analyzed (n= 5-10). (B) Proliferating TIL-MDSCs in tumor bearing mice were analyzed as BrdU⁺CD11b⁺Gr-1⁺ cells 24 hours post-BrdU injection. (C) Representative tumor histology images from WT and p53^null mice. Scale bar = 100 mm. (D) Representative tumor IHC images (200X) from WT and p53^null mice revealing blood endothelial cells (BEC, LYVE-1⁻Gp38⁻CD31⁺), lymphatic endothelial cells (LEC, LYVE-1⁺Gp38⁺CD31⁺), fibroblastic reticular cells (FRC, ER-TR7⁺Gp38⁺α-SMA⁺), and CD11b⁺ cells. The relative area of CD31⁺ vasculatures and ER-TR7⁺ FRC-stroma was determined using a computer-assisted program. Data are mean ± s.e. (n= 3). * denotes a significant difference (p < 0.05).

Fig. 3. B16F1 establishment in p53^null hosts promotes a systemic increase in proinflammatory cytokines, MDSCs, and stromal-network in the spleen

(A) Serum proinflammatory cytokines/chemokines in tumor bearing mice were determined via cytokine multiplex array (n=6). (B) The splenic MDSCs in tumor bearing WT and p53^null mice were analyzed. (C) Proliferating splenic MDSCs in tumor bearing mice were examined 24 hours post-BrdU injection. (n=3). (D) Representative splenic IHC images (200X) of non-tumor and tumor bearing mice. * denotes a significant difference (p < 0.05) between WT and p53^null mice under the same treatment, whereas † indicates a significant difference between non-tumor and tumor bearing mice.

Fig. 4. Trp53^null splenic fibroblastic stroma enhances MDSC differentiation and accelerates tumor progression in WT mice

(A) A representative image (400X) and phenotypic analyses of CD45⁻ splenic stromal cells (SPSC) from tumor bearing p53^null mice. Open lines indicate isotype controls. (B) BM-MDSCs were differentiated in a 4-day culture with G-CSF and IL-6 with or without SPSCs. Their relative efficiency was normalized against that of WT BM-MDSCs without SPSCs, which was set as 100%. Proliferating (BrdU⁺) BM-MDSCs were examined o day 4 (n=3). * denotes a significant difference (p < 0.05) between groups with and without SPSCs, whereas † indicates a significant difference between WT and p53^null BM-MDSCs. (C) Proinflammatory molecule expression in p53^null SPSCs compared with those in MEF, as well as their alterations upon a 6-hour exposure to B16F1-conditioned medium, was determined via real-time RT-PCR (n=3-5). (D) The tumor size in WT mice received 2 × 10⁵ B16F1 or 2 × 10⁵ B16F1 admixed with 1 × 10⁵ p53^null SPSCs was compared (n=5). * denotes a significant difference (p < 0.05).