Tumor Growth In Wild-type Mice Research Articles

Abstract 2465: A novel agonist of CD137 as a single agent shows cancer immunoprevention efficacy against various tumor types that is not dependent on the trained immunity

Abstract Introduction: We recently demonstrated that pretreatment of mice with SA-4-1BBL, a novel oligomeric form of CD137 agonist, as a single agent prevents the development of various tumor types in mice. The tumor immunoprevention efficacy is long-lasting (>14 weeks) and contingent upon CD4+ immune cells, NK cells, and IFN-γ, but not CD8+ T and B cells. Trained immunity mediated by previously activated myeloid cells has been shown to have efficacy against cancer. The objective of this study was twofold: i) assess the role of trained immunity and ii) elucidate the mechanistic underlying of cancer immunoprevention. Study Design: Wild-type C57BL/6 mice and various transgenic (CD4+ OT-II.Rag+/+, CD4+ OT-II.Rag−/−, CD8+ OT-I/Rag2−/- KO, and Rag1−/−) mice on C57BL/6 background were pretreated subcutaneously with SA-4-1BBL or an agonistic Ab that does not have immunoprevention efficacy (3H3) twice two weeks apart. Mice were challenged two weeks later with a cervical cancer cell line (TC-1) and monitored for tumor development. A group of mice was also treated with monophosphoryl lipid A (MPLA), an agonist of TLR4 involved in trained immunity, using the above scheme. Mice treated with saline served as the control throughout all studies. Deep immunophenotyping was conducted to assess immune correlates of cancer immunoprevention. Results: Pretreatment with SA-4-1BBL prevented tumor growth in wild-type mice (> 60%), whereas all MPLA treated mice developed tumor. SA-4-1BBL did not show tumor immunoprevention efficacy in mice lacking adaptive immune cells (Rag1−/- KO), OT-II cells on Rag−/- or Rag+/+ background, and OT-I on Rag2−/− background. Deep immunophenotyping revealed a significant increase in the absolute number of CD4+ T central memory and CD4+ T stem cell-like memory cells, NK cells, NKT cells, and various myeloid cells in mice treated with SA-4-1BBL as compared with those treated with the 3H3 Ab or saline treated controls. Conclusions: These results demonstrate that cancer immunoprevention efficacy of SA-4-1BBL requires a diverse T cell receptor repertoire as OT-II mice on Rag+/+ background, restricted to a TCRβ chain, but have all other myeloid and NK cells, do not show efficacy. Furthermore, treatment with MPLA involved in trained immunity has no efficacy. Altogether, these observations do not implicate trained immunity as a mechanism of cancer immunoprevention. Elucidation of the mechanistic basis of SA-4-1BBL immunoprevention may have significant clinical implications. Citation Format: Feyza Nur Arguc, Mohammad Tarique, Vahap Ulker, Esma S. Yolcu, Haval Shirwan. A novel agonist of CD137 as a single agent shows cancer immunoprevention efficacy against various tumor types that is not dependent on the trained immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2465.

IL1α Antagonizes IL1β and Promotes Adaptive Immune Rejection of Malignant Tumors.

We assessed the contribution of IL1 signaling molecules to malignant tumor growth using IL1β-/-, IL1α-/-, and IL1R1-/- mice. Tumors grew progressively in IL1R-/- and IL1α-/- mice but were often absent in IL1β-/- mice. This was observed whether tumors were implanted intradermally or injected intravenously and was true across multiple distinct tumor lineages. Antibodies to IL1β prevented tumor growth in wild-type (WT) mice but not in IL1R1-/- or IL1α-/- mice. Antibodies to IL1α promoted tumor growth in IL1β-/- mice and reversed the tumor-suppressive effect of anti-IL1β in WT mice. Depletion of CD8+ T cells and blockade of lymphocyte mobilization abrogated the IL1β-/- tumor suppressive effect, as did crossing IL1β-/- mice to SCID or Rag1-/- mice. Finally, blockade of IL1β synergized with blockade of PD-1 to inhibit tumor growth in WT mice. These results suggest that IL1β promotes tumor growth, whereas IL1α inhibits tumor growth by enhancing T-cell-mediated antitumor immunity.

Complex interrelationships between nitro-alkene-dependent inhibition of soluble epoxide hydrolase, inflammation and tumor growth

Nitro-oleate (10-nitro-octadec-9-enoic acid), which inhibits soluble epoxide hydrolase (sEH) by covalently adducting to C521, increases the abundance of epoxyeicosatrienoic acids (EETs) that can be health promoting, for example by lowering blood pressure or their anti-inflammatory actions. However, perhaps consistent with their impact on angiogenesis, increases in EETs may exacerbate progression of some cancers. To assess this, Lewis lung carcinoma (LLc1) cells were exposed to oleate or nitro-oleate, with the latter inhibiting the hydrolase and increasing their proliferation and migration in vitro. The enhanced proliferation induced by nitro-oleate was EET-dependent, being attenuated by the ETT-receptor antagonist 14,15-EE-5(Z)-E. LLc1 cells were engineered to stably overexpress wild-type or C521S sEH, with the latter exhibiting resistance to nitro-oleate-dependent hydrolase inhibition and the associated stimulation of tumor growth in vitro or in vivo. Nitro-oleate also increased migration in endothelial cells isolated from wild-type (WT) mice, but not those from C521S sEH knock-in (KI) transgenic mice genetically modified to render the hydrolase electrophile-resistant. These observations were consistent with nitro-oleate promoting cancer progression, and so the impact of this electrophile was examined in vivo again, but this time comparing growth of LLc1 cells expressing constitutive levels of wild-type hydrolase when implanted into WT or KI mice. Nitro-oleate inhibited tumor sEH (P < 0.05), with a trend for elevated plasma 11(12)-EET/DHET and 8(9)EET/DHET (dihydroxyeicosatrienoic acid) ratios when administered to WT, but not KI, mice. Although in vitro studies with LLc1 cells supported a role for nitro-oleate in cancer cell proliferation, it failed to significantly stimulate tumor growth in WT mice implanted with the same LLc1 cells in vivo, perhaps due to its well-established anti-inflammatory actions. Indeed, pro-inflammatory cytokines were significantly down-regulated in nitro-oleate treated WT mice, potentially countering any impact of the concomitant inhibition of sEH.

Abstract A062: TIM-3 plays distinct roles in different immune cells to regulate antitumor immune responses

Abstract T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) was originally identified as an inhibitory receptor that is expressed on Th1 T-cells to dampen T-cell immunity upon interaction with several putative ligands. The inhibitory role of TIM-3 is supported by multiple preclinical disease models, such as viral and bacterial infections, experimental autoimmune encephalitis, and allograft rejection. Recent evidence suggests that induction of TIM-3 expression on T-cells may promote resistance to cancer immunotherapy, including in response to treatment with PD-1/PD-L1 inhibitors. Thus, TIM-3 represents a putative novel immuno-oncology target. Here we report that in naïve mice TIM-3 is absent on T-cells, but is constitutively expressed on myeloid cells, including dendritic cells and macrophages. In tumor-bearing mice, TIM-3 expression is highly enriched on PD-1 positive CD4 and CD8 T-cells in the tumor microenvironment, but not on T-cells in draining lymph nodes or peripheral blood. Prophylactic or therapeutic treatment with anti-PD-1 blocking antibody delays tumor growth in wild-type mice. However, unexpectedly, TIM-3 genetic deficiency reduced overall survival of tumor-bearing mice treated with anti-PD-1 compared to wild-type control mice. Using a series of in vitro functional cell-based assays, we found that blocking TIM-3 function by either genetic knock-out or an inhibitory Ab increased proliferation of, and IFN-γ production by, effector CD8 T-cells following direct antigen stimulation. By contrast, TIM-3 gene knockout in bone marrow-derived macrophages did not impact responses to stimulation with Toll-like receptor ligands, and TIM-3 blockade reduced phagocytosis of apoptotic tumor cells by a macrophage cell line. Taken together, our results suggest TIM-3 may play opposite roles in T-cells and macrophages (inhibitory vs activating, respectively), and highlight the pleiotropic roles of TIM-3 in different immune cells in tumor immunology. Citation Format: Jie Dai, Jerry Pei, Markus Mohrs, Gavin Thurston, Ella Ioffe. TIM-3 plays distinct roles in different immune cells to regulate antitumor immune responses [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A062.

Abstract 4727: MicroRNA-155 as a regulator of NK cell mediated solid tumor rejection

Abstract Natural Killer (NK) cells are granular innate lymphocytes that orchestrate immune response by releasing interferon gamma (IFN-γ): a critical cytokine heavily involved in intercellular immune and inflammatory processes. Prior work from our lab has linked microRNA-155 (miR-155) to significant cell proliferation and NK cell mediated IFN-γ release. Previously, our lab has also shown that miR-155 can increase hematological malignancy rejection and NK cell cytotoxicity in T-cell lymphomas. However, it is unclear how miR-155 can affect NK cell cytotoxicity in solid tumor models. IFN-γ has been reported in the literature to directly induce apoptosis in glioblastoma solid tumors. We therefore hypothesize that miR-155 induction can increase NK cell mediated cytotoxicity and IFN-γ mediated apoptosis for solid tumor rejection. To test this, 10x106 GL261 cells were injected subcutaneously into the flanks of both wild type (WT) littermates and miR-155 transgenic (miR-155Tg) mice. CD3(-)NK1.1(+) effector splenic NK cells were isolated from WT and miR-155Tg mice for total RNA extraction and subsequent gene expression profiling on NK cell activation pathways. We found that tumor growth in WT mice was at least 2-fold greater than that of miR-155Tg mice (average 2cmx2cm vs 0.7cmx0.7cm, n=6). Tumor weights from WT and miR-155 mice were 1052 mg vs 340 mg, respectively, with an average of 2 mice per group. Furthermore, we have successfully created an IFN-γ receptor 1 (IFN-γR1) shRNA knock-down (KD) lentiviral construct in a stable glioblastoma cell line. IFN-γR1 knockdown was measured to be 90% as determined by real-time PCR analysis. Future experiments will be performed wherein IFN-γR1 KD glioblastoma cells will be injected in miR-155Tg and WT mice to evaluate tumor rejection. In summary, we demonstrate a model system to investigate the effect of miR-155 on solid tumor glioblastoma multiforme. Further characterization of gene expression profiles of purified NK cells from both WT and miR-155 mice will provide important insights on the mechanisms of NK cell medicated tumor rejection. Citation Format: Aman Prasad, Wing Keung Chan. MicroRNA-155 as a regulator of NK cell mediated solid tumor rejection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4727.

Peritumoral IL-15 Promotes Regional NK Activation and DC Licensing to Augment Radiation-Induced Anti-tumor Immunity

Radiotherapy (RT) can elicit anti-tumor T cells by multiple mechanisms but the induced immune response is limited by the immunosuppression in the tumor microenvironment. The common gamma-chain cytokines IL-15 promotes the activation of natural killer (NK) cells and the proliferation of activated CD8+ T cells and is under investigation for its ability to induce T cell response in cancer patients. Here we tested the hypothesis that IL-15 strengthens the pro-immunogenic effect of local RT to potentiate durable tumor-specific immunity. The poorly immunogenic mouse TSA breast cancer cells were implanted s.c. in syngeneic wild type (WT) or batf3-deficient BALB/c mice and when tumors reached 5mm in average diameter, were randomly assigned to one of 4 treatment groups: control, RT, IL-15 or RT+IL-15. Local RT was delivered consecutively in 8 Gy fractions on days 13, 14, and 15 post tumor implantation. IL-15 (2 μg/mouse) was administered s.c. peritumorally daily for 10 days starting on the first day of RT. Mice were followed for tumor growth and surviving mice subsequently re-challenged. Some animals were depleted of CD8 and NK cells (anti-asialo-GM1 Ab) using standard protocols. A parallel experiment was done to characterize tumor-infiltrating lymphocytes (TILs) and measure ex vivo IFNg production by tumor-specific CD8 T-cells in the draining lymph nodes at the end of treatment (day 22). IL-15 as monotherapy had no effect on tumor control but significantly improved the ability of RT to delay tumor growth in WT mice (P < 0.01, RT vs RT+IL-15). Combination treatment led to durable and robust memory responses in 50% of animals compared to 0% of mice treated with RT alone. RT enhanced intratumoral infiltration of DX5+NKp46+ NK cells and was further increased by the addition of IL-15 (22.86% of CD45 TILs in RT vs 32% in RT+IL-15, P<0.05). CD8+ T cells expressing the activation marker CD137 were significantly increased in tumors of RT+IL-15-treated mice while the increase was modest with each monotherapy (35.3% in RT+IL-15 vs 18.8% in RT, 20.7% in IL-15, 5.90% in control, P < 0.05). Systemic depletion of either CD8 T-cells or NK cells abrogated the therapeutic effect of the combination. RT+IL-15 elicited more priming of tumor-specific T-cells in response to tumor epitope AH-1-A5 (1238 pg/mL in RT+IL-15 vs 3.3 pg/mL in control vs 28.7 pg/mL in IL-15 RT vs 2.63 pg/mL in RT; P<0.05). Batf3-/- mice that CD103+ DCs showed reduce response to RT+IL-15 compared to WT mice. Here we show that local IL-15 administration improves priming of anti-tumor CD8+ T cell and leads to long-term memory responses by a mechanism that is dependent on NK cells and Batf3-dependent DCs. Data suggest that IL-15 activated NK cells kill tumor cells and license DCs leading to a long-term T cell-mediated memory response. Overall these results support the testing of IL-15 in combination with tumor-targeted radiotherapy.

Intestinal microbiota alters omental tumor growth (TUM9P.1004)

Abstract The omentum is an adipose tissue that contains milky spots (MS). The MS are similar to secondary lymphoid organs and can generate B and T cell immune responses to peritoneal antigens. Additionally, omentum CD4+CD25+FoxP3+ T regulatory cells (Tregs) display an activated phenotype CD44hiCD62Llow. Moreover, the omentum also collects metastasizing tumor cells in the peritoneal cavity and tumors growing in the omentum are associated with poor clinical prognosis. Here we show that intestinal microbiota affects Tregs activation profile and impairs tumor growth in omentum. Our transplantable tumor model in wild type (WT) mice shows that peritoneal tumors grow progressively in the omentum and peritoneal cavity in WT mice, whereas in germ free (GF) mice tumors do not grow. Omental tumor growth in WT mice is associated with an increase in PD-1+ Tregs, while tumor-specific CD8+ T are reduced. However, in GF mice numbers of PD1+ Tregs remain unaltered but tumor specific CD8+ T cells are still present. After co-housed GF mice with WT mice, ex-GF mice showed tumor growth and restore the increase in PD-1+ Tregs with low number of specific CD8+ T cells. The induction of tolerance requires PD-1+ Tregs. These data suggest that intestinal microbiota has a role in omental Tr activation and in the tolerance to peritoneal tumors.

Abstract 1548: Ultraviolet B-induced platelet-activating factor receptor agonists mediate augmentation of melanoma tumor growth via regulatory T cells: Implication of systemic immunosuppression

Abstract Ultraviolet B (UVB)-irradiation to human or mouse skin generates Platelet-activating factor (PAF) and novel oxidatively-modified glycerophosphocholines (Ox-GPCs) with PAF-receptor (PAF-R) agonistic activity. These lipids mediate systemic immunosuppression in a process involving IL-10. The current studies sought to determine the functional significance of UVB-mediated systemic immunosuppression in an established model of murine melanoma progression. As our studies demonstrated that B16F10 tumors do not express functional PAF-Rs, retroviral-mediated transduction of the PAF-R was used to create PAF-R-positive B16F10 melanoma tumor cells. We show that UVB irradiation augments B16F10 tumor growth and is dependent on host, but not melanoma cell, PAF-R-expression as UVB or the PAF-R agonist, carbamoyl- PAF (CPAF), both promote B16F10 tumor growth in wild-type mice, independent of whether B16F10 cells express PAF-Rs, but do not augment tumor growth in PAF-R deficient (Pafr -/-) mice. Systemic antioxidant treatment ameliorated UVB-mediated augmentation of tumor growth, indicating involvement of Ox-GPC PAF-R agonists produced non-enzymatically. Host immune cells are required as CPAF-induced augmentation of tumor growth is not seen in immunodeficient NOD SCID mice. Finally, depleting antibodies against IL-10 in WT mice, or depletion of CD25-positive cells (suggestive of FoxP3 directed regulatory T cells) in FoxP3EGFP transgenic mice block UVB/CPAF-induced tumor growth, supporting a requirement for IL-10 and regulatory T cells (Tregs) in this process. These findings indicate that UVB-generated Ox-GPCs with PAF-R agonistic activity enhance experimental murine melanoma tumor growth through targeting host immune cells, most notably Tregs, to mediate systemic immunosuppression. These studies describe a previously unrecognized mechanism by which melanoma tumor cells can escape antitumor immunity. Inasmuch as other pro-oxidative stressors could potentially generate PAF-R agonists, this pathway could have clinical relevance in terms of devising better strategy for melanoma immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1548. doi:1538-7445.AM2012-1548

Host-Derived MCP-1 and MIP-1α Regulate Protective Anti-Tumor Immunity to Localized and Metastatic B16 Melanoma

Leukocytic infiltration into malignant melanoma lesions is tightly regulated by chemokines. To assess the role of the CC chemokines monocyte chemotactic protein-1 (MCP-1/chemokine ligand 2) and macrophage inflammatory protein-1α (MIP-1α/chemokine ligand 3) in this process, s.c. primary and metastatic B16 F10 melanoma tumor growth levels were examined in mice lacking MCP-1 or MIP-1α. Primary s.c. B16 F10 melanoma growth was augmented by loss of MCP-1 or MIP-1α. Similarly, lung metastasis was enhanced by the deficiency of MCP-1 or MIP-1α. Enhanced tumor outgrowth was associated with decreased percentages of infiltrating CD4(+) T cells, CD8(+) T cells, and natural killer cells. In the absence of MCP-1 or MIP-1α, melanoma outgrowth was correlated with reduced local expression of interferon-γ, IL-6, tumor necrosis factor-α, and transforming growth factor-β. Among these cytokines, reduced expression levels of interferon-γ and tumor necrosis factor-α on leukocytes from the spleen were associated with the development of lung metastasis in chemokine-deficient mice. The local s.c. administration of these four cytokines significantly augmented another chemokine's expression and suppressed primary melanoma growth in mice deficient for MCP-1 or MIP-1α. The s.c. injection of MCP-1 or MIP-1α significantly inhibited the primary tumor growth in wild-type mice. These results indicate that host-derived MCP-1 and MIP-1α regulate protective anti-tumor immunity to B16 F10 melanoma by promoting lymphocyte infiltration into the tumor and subsequent cytokine production.

Lymphotoxin‐β receptor activation by lymphotoxin‐α1β2 and LIGHT promotes tumor growth in an NFκB‐dependent manner

Lymphotoxin beta receptor (LTβR) activation on mouse fibrosarcoma cells (BFS-1) results in enhanced solid tumor growth paralleled by increased angiogenesis induced by the expression of pro-angiogenic CXCL2. In our study, we demonstrate that both functional ligands of the LTβR, namely LTα(1) β(2) and LIGHT, are involved in the activation of LTβR in solid fibrosarcomas. To identify whether the lymphocyte population is involved in the activation of LTβR in these fibrosarcoma tumors, we used conditional LTβ-deficient mice that specifically lack LTβ expression either on T cells (T-LTβ(-/-)) or on B cells (B-LTβ(-/-)). Solid tumor growth was reduced in both mouse strains when compared to tumor growth in wild-type mice, indicating the participation of both T and B host lymphocytes in the activation of LTβR in these tumors. Tumor growth was also reduced in LIGHT-deficient mice, suggesting a contribution of this ligand to the activation of LTβR in BFS-1 fibrosarcomas. LTβR signaling can involve IκBα and/or NFκB-inducing kinase (NIK) for subsequent NFκB activation in different types of cells. Expression of a dominant negative form of IκBα or of a dominant negative mutant of NIK resulted in decreased activation of NFκB signaling and reduced expression of pro-angiogenic CXCL2 in vitro. Moreover, expression of dominant negative form of NIK or an IκBα repressor in these fibrosarcoma cells resulted in reduced solid tumor growth in vivo, suggesting that both IκBα and NIK are involved in pro-angiogenic signaling after LTβR activation. Our data support the idea that the ablation of LTβR signaling should be considered for cancer treatment.

Local Toll-like receptor-mediated mast cell activation inhibits melanoma growth

Mast cells are abundant at the periphery of the majority of solid tumors where they typically promote angiogenesis and enhance tumor growth. However, there is some evidence from epidemiological studies that allergic disease reduces cancer risk. In the context of infection, mast cell activation with Toll-like receptor (TLR) agonists induces the production of multiple mediators which mobilize and activate known anti-tumor effector cells. TLR agonists can also be effective in tumor immunotherapy. We demonstrate that the TLR2 agonist Pam3CSK4 is tumor inhibitory and mediates its effects via mast cell activation and reversal of the normal pro-tumorigenic activity of mast cells. TLR2 agonist treatment decreased B16.F10 tumor growth in wild-type mice, but not in mast cell deficient Kit W-sh/W-sh mice. Tumor growth inhibition following TLR2 agonist administration was restored in Kit W-sh/W-sh mice by local reconstitution with wild-type, but not TLR2-deficient mast cells. Mast cell dependent tumor growth inhibition occurred independently of tumor necrosis factor (TNF) production and was not associated with direct tumor cytotoxicity or degranulation in vitro. Tumor growth inhibition was associated with mast cell dependent recruitment of natural killer (NK) and T cells. This study demonstrates that local mast cell activation provides a novel opportunity to modify the tumor microenvironment for successful cancer immunotherapy. Supported by the CCSRI and CIHR. S.A.O is a recipient of support from the Nova Scotia Health Research Foundation.

IL-17 Promotes Tumor Development through the Induction of Tumor Promoting Microenvironments at Tumor Sites and Myeloid-Derived Suppressor Cells

The role of immune responses in tumor development is a central issue for tumor biology and immunology. IL-17 is an important cytokine for inflammatory and autoimmune diseases. Although IL-17-producing cells are detected in cancer patients and tumor-bearing mice, the role of IL-17 in tumor development is controversial, and mechanisms remain to be fully elucidated. In the current study, we found that the development of tumors was inhibited in IL-17R-deficient mice. A defect in IFN-gammaR increased tumor growth, whereas tumor growth was inhibited in mice that were deficient in both IL-17R and IFN-gammaR compared with wild-type animals. Further experiments showed that neutralization of IL-17 by Abs inhibited tumor growth in wild-type mice, whereas systemic administration of IL-17 promoted tumor growth. The IL-17R deficiency increased CD8 T cell infiltration, whereas it reduced the infiltration of myeloid-derived suppressor cells (MDSCs) in tumors. In contrast, administration of IL-17 inhibited CD8 T cell infiltration and increased MDSCs in tumors. Further analysis indicated that IL-17 was required for the development and tumor-promoting activity of MDSCs in tumor-bearing mice. These data demonstrate that IL-17-mediated responses promote tumor development through the induction of tumor-promoting microenvironments at tumor sites. IL-17-mediated regulation of MDSCs is a primary mechanism for its tumor-promoting effects. The study provides novel insights into the role of IL-17 in tumor development and has major implications for targeting IL-17 in treatment of tumors.

Function of endogenous inhibitors of angiogenesis as endothelium-specific tumor suppressors

Disruption of the systemic angiogenesis balance to favor enhanced angiogenesis is speculated to represent a key step in the growth of tumors. Although a major emphasis has been placed on the increase of angiogenesis stimulators, such as VEGF, on the disruption of the angiogenic balance, the potential role of the physiological levels of endogenous inhibitors of angiogenesis on tumor growth is poorly understood. Here, we use three independent lines of mice deficient in tumstatin, endostatin, or thrombospondin-1 (TSP-1), to address the role that these endogenous angiogenesis inhibitors play in tumor growth. Our experiments demonstrate that normal physiological levels of these inhibitors serve to retard the growth of tumors, and that their absence leads to enhanced angiogenesis and a 2- to 3-fold increase in tumor growth. The tumor-suppressive action of TSP-1, endostatin, and tumstatin correlates with expression of CD36 receptor, alpha5beta1 integrin, and alphavbeta3 integrin on proliferating endothelial cells, respectively. Moreover, tumors grow 2-fold faster in the tumstatin/TSP-1 double-knockout mice, compared with either the tumstatin- or the TSP-1-deficient mice, strongly suggesting that ceiling rate of cancer growth is not completely dependent on the genetic defects of cancer cells but also depends on the host-derived tumor microenvironment. Additionally, tumor growth in transgenic mice overproducing endostatin specifically in the endothelial cells (a 1.6-fold increase in the circulating levels; mimicking Down's syndrome condition) is 3-fold slower than the tumor growth in wild-type mice. Collectively, our data suggest that physiological levels of endogenous inhibitors of angiogenesis can serve as endothelium-specific tumor suppressors.