Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Apr;61(4):511-21.
doi: 10.1007/s00262-011-1119-y. Epub 2011 Oct 5.

The small molecule TGF-β signaling inhibitor SM16 synergizes with agonistic OX40 antibody to suppress established mammary tumors and reduce spontaneous metastasis

Kendra Garrison  1 , Tobias Hahn, Wen-Cherng Lee, Leona E Ling, Andrew D Weinberg, Emmanuel T Akporiaye
Affiliations

The small molecule TGF-β signaling inhibitor SM16 synergizes with agonistic OX40 antibody to suppress established mammary tumors and reduce spontaneous metastasis

Kendra Garrison et al. Cancer Immunol Immunother. 2012 Apr.

Abstract

Effective tumor immunotherapy may require not only activation of anti-tumor effector cells, but also abrogation of tumor-mediated immunosuppression. The cytokine TGF-β, is frequently elevated in the tumor microenvironment and is a potent immunosuppressive agent and promoter of tumor metastasis. OX40 (CD134) is a member of the TNF-α receptor superfamily and ligation by agonistic antibody (anti-OX40) enhances effector function, expansion, and survival of activated T cells. In this study, we examined the therapeutic efficacy and anti-tumor immune response induced by the combination of a small molecule TGF-β signaling inhibitor, SM16, plus anti-OX40 in the poorly immunogenic, highly metastatic, TGF-β-secreting 4T1 mammary tumor model. Our data show that SM16 and anti-OX40 mutually enhanced each other to elicit a potent anti-tumor effect against established primary tumors, with a 79% reduction in tumor size, a 95% reduction in the number of metastatic lung nodules, and a cure rate of 38%. This positive treatment outcome was associated with a 3.2-fold increase of tumor-infiltrating, activated CD8+ T cells, an overall accumulation of CD4+ and CD8+ T cells, and an increased tumor-specific effector T cell response. Complete abrogation of the therapeutic effect in vivo following depletion of CD4+ and CD8+ T cells suggests that the anti-tumor efficacy of SM16+ anti-OX40 therapy is T cell dependent. Mice that were cured of their tumors were able to reject tumor re-challenge and manifested a significant tumor-specific peripheral memory IFN-γ response. Taken together, these data suggest that combining a TGF-β signaling inhibitor with anti-OX40 is a viable approach for treating metastatic breast cancer.

PubMed Disclaimer

Conflict of interest statement

The authors report no conflict of interest.

Figures

Fig. 1
Fig. 1
SM16 plus anti-OX40 combination therapy suppresses growth of established mammary tumors and spontaneous metastases. Mice were injected in the mammary pad with 5 × 104 4T1 tumor cells. On day 10 when tumors were palpable (~22mm2), mice were randomized to the control and various treatment groups. Mice received 3 injections of OX40 (250 μg/injection/mouse) or isotype control antibody at 3-day intervals beginning on day 15 after tumor establishment when the average tumor size in all groups was ~40mm2. a Individual tumor sizes; numbers in parentheses represent mean ± SEM of tumor areas. b Tumor growth kinetics of individual mice. The number of mice per treatment group (N = 31–32) is combined from 4 independent experiments. c On day 28 post-tumor cell injection, untreated mice and mice treated with SM16, anti-OX40, or both were culled and visible lung metastases were enumerated. Number of visible lung metastases; numbers in parentheses represent mean ± SEM combined from 3 independent experiments. d Relationship between tumor size and frequency of visible lung metastases
Fig. 2
Fig. 2
SM16+ anti-OX40 treatment increases the number of activated T cells in the tumor microenvironment. Mice with established 10-day tumors received 0.9 mg SM16/mouse/day orally in the diet for 18 days and were each given 3 separate injections of anti-OX40 on days 15, 18, and 21 post-tumor injection. On day 28, tumor-infiltrating immune cells (TIC) were isolated and analyzed by polychromatic flow cytometry. a Gating strategy of tumor-infiltrating, activated CD4+ and CD8+ T cells. b Frequency of total and activated CD8+ and CD4+ T cells. Activated T cells were identified as CD4+CD44+CD62L− or CD8+CD44+CD62L−; c Gating strategy of CD4+ CD25+ Foxp3+ Treg. d Frequency of CD4+ CD25+ Foxp3+ Treg. TICs were analyzed from 4 individual mice per group in 3 independent experiments. The data represent mean ± SEM of 12 mice/group. Dead cells were excluded for all analyses by viability stain. Asterisk indicates significant difference between SM16 + anti-OX40 group and the control, SM16 and anti-OX40 groups (P < 0.05)
Fig. 3
Fig. 3
Effect of SM16+ anti-OX40 therapy on cytokine production. Draining inguinal lymph nodes were harvested and pooled from tumor-bearing mice (N = 3/group) on day 24 post-tumor cell injection. The isolated cells were incubated with irradiated 4T1 or Renca tumor cells for 48 h. Supernatants were collected and assessed for a IFN-γ (asterisk indicates significant difference between the SM16+ anti-OX40 group and the control group) and b IL-4 (asterisk indicates significant difference between the control group and all other groups) secretion by ELISA. Data represent IFN-γ and IL-4 levels after subtracting cytokine levels in Renca-stimulated cultures from 4T1-stimulated cultures
Fig. 4
Fig. 4
Depletion of T cells abrogates efficacy of SM16+ anti-OX40 therapy. Mice with 8-day established 4T1 mammary tumors (N = 3/group) were depleted of T cells by weekly intra-peritoneal injections of CD4- and/or CD8-specific antibodies prior to (day 8) and during SM16+ anti-OX40 therapy (day 15 and day 22 post-tumor implantation). A cohort of T cell replete mice received matched isotype antibody and SM16+ anti-OX40 treatment. a Tumor growth was monitored weekly (asterisk indicates significant difference between anti-CD4 and anti-CD8 group and all other treatment groups). b Mice in all groups were culled when tumor size in the control group reached ~200 mm2 and number of lung metastases were determined (asterisk indicates significant difference between anti-CD4 and anti-CD8 group and all other treatment groups)
Fig. 5
Fig. 5
SM16+ anti-OX40 treatment generates a tumor-specific memory response. a Schematic representation of treatment protocol. Mice bearing established 4T1 tumors were started on SM16 diet on day 10 followed by 3 anti-OX40 injections on days 15, 18, and 21. SM16 diet was discontinued on day 28 and tumor-free mice were challenged with 5 × 104 4T1 and 5 × 105 Renca cells tumor cells in opposite flanks. Tumor growth was monitored for up to 21 days (day 80). b Draining inguinal lymph nodes were harvested from SM16+ anti-OX40-treated mice 21 days after 4T1 tumor re-challenge and isolated cells were incubated for 48 h with irradiated (300 Gy) 4T1 or Renca tumor cells. Supernatant fluid was collected and assessed for IFN-γ secretion by ELISA
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Huddleston CA, Weinberg AD, Parker DC. OX40 (CD134) engagement drives differentiation of CD4 + T cells to effector cells. Eur J Immunol. 2006;36(5):1093–1103. doi: 10.1002/eji.200535637. - DOI - PubMed
    1. Krasagakis K, Tholke D, Farthmann B, Eberle J, Mansmann U, Orfanos CE. Elevated plasma levels of transforming growth factor (TGF)-beta1 and TGF-beta2 in patients with disseminated malignant melanoma. Br J Cancer. 1998;77(9):1492–1494. doi: 10.1038/bjc.1998.245. - DOI - PMC - PubMed
    1. Bellone G, Turletti A, Artusio E, Mareschi K, Carbone A, Tibaudi D, Robecchi A, Emanuelli G, Rodeck U. Tumor-associated transforming growth factor-beta and interleukin-10 contribute to a systemic Th2 immune phenotype in pancreatic carcinoma patients. Am J Pathol. 1999;155(2):537–547. doi: 10.1016/S0002-9440(10)65149-8. - DOI - PMC - PubMed
    1. Shim KS, Kim KH, Han WS, Park EB. Elevated serum levels of transforming growth factor-beta1 in patients with colorectal carcinoma: its association with tumor progression and its significant decrease after curative surgical resection. Cancer. 1999;85(3):554–561. doi: 10.1002/(SICI)1097-0142(19990201)85:3<554::AID-CNCR6>3.0.CO;2-X. - DOI - PubMed
    1. Kong FM, Anscher MS, Murase T, Abbott BD, Iglehart JD, Jirtle RL. Elevated plasma transforming growth factor-beta 1 levels in breast cancer patients decrease after surgical removal of the tumor. Ann Surg. 1995;222(2):155–162. doi: 10.1097/00000658-199508000-00007. - DOI - PMC - PubMed

Publication types

MeSH terms