The cardioprotective protein apolipoprotein A1 promotes potent anti-tumorigenic effects.

Here, we show that apolipoprotein A1 (apoA1), the major protein component of high density lipoprotein (HDL), through both innate and adaptive immune processes, potently suppresses tumor growth and metastasis in multiple animal tumor models, including the aggressive B16F10L murine malignant melanoma model. Mice expressing the human apoA1 transgene (A1Tg) exhibited increased infiltration of CD11b(+) F4/80(+) macrophages with M1, anti-tumor phenotype, reduced tumor burden and metastasis, and enhanced survival. In contrast, apoA1-deficient (A1KO) mice showed markedly heightened tumor growth and reduced survival. Injection of human apoA1 into A1KO mice inoculated with tumor cells remarkably reduced both tumor growth and metastasis, enhanced survival, and promoted regression of both tumor and metastasis burden when administered following palpable tumor formation and metastasis development. Studies with apolipoprotein A2 revealed the anti-cancer therapeutic effect was specific to apoA1. In vitro studies ruled out substantial direct suppressive effects by apoA1 or HDL on tumor cells. Animal models defective in different aspects of immunity revealed both innate and adaptive arms of immunity contribute to complete apoA1 anti-tumor activity. This study reveals a potent immunomodulatory role for apoA1 in the tumor microenvironment, altering tumor-associated macrophages from a pro-tumor M2 to an anti-tumor M1 phenotype. Use of apoA1 to redirect in vivo elicited tumor-infiltrating macrophages toward tumor rejection may hold benefit as a potential cancer therapeutic.

Combination of vitamin D and photodynamic therapy enhances immune responses in murine models of squamous cell skin cancer.

Improved treatment outcomes for non-melanoma skin cancers can be achieved if Vitamin D (Vit D) is used as a neoadjuvant prior to photodynamic therapy (PDT). However, the mechanisms for this effect are unclear. Vit D elevates protoporphyrin (PpIX) levels within tumor cells, but also exerts immune-modulatory effects. Here, two murine models, UVB-induced actinic keratoses (AK) and human squamous cell carcinoma (A431) xenografts, were used to analyze the time course of local and systemic immune responses after PDT ± Vit D. Fluorescence immunohistochemistry of tissues and flow analysis (FACS) of blood were employed. In tissue, damage-associated molecular patterns (DAMPs) were increased, and infiltration of neutrophils (Ly6G+), macrophages (F4/80+), and dendritic cells (CD11c+) were observed. In most cases, Vit D alone or PDT alone increased cell recruitment, but Vit D + PDT showed even greater recruitment effects. Similarly for T cells, increased infiltration of total (CD3+), cytotoxic (CD8+) and regulatory (FoxP3+) T-cells was observed after Vit D or PDT, but the increase was even greater with the combination. FACS analysis revealed a variety of interesting changes in circulating immune cell levels. In particular, neutrophils decreased in the blood after Vit D, consistent with migration of neutrophils into AK lesions. Levels of cells expressing the PD-1+ checkpoint receptor were reduced in AKs following Vit D, potentially counteracting PD-1+ elevations seen after PDT alone. In summary, Vit D and ALA-PDT, two treatments with individual immunogenic effects, may be advantageous in combination to improve treatment efficacy and management of AK in the dermatology clinic.

Healthy myeloid-derived suppressor cells express the surface ectoenzyme Vanin-2 (VNN2).

Study of human monocytic Myeloid-Derived Suppressor cells Mo-MDSC (CD14+ HLA-DRneg/low) has been hampered by the lack of positive cell-surface markers. In order to identify positive markers for Mo-MDSC, we performed microarray analysis comparing Mo-MDSC cells from healthy subjects versus CD14+ HLA-DRhigh monocytes. We have identified the surface ectoenzyme Vanin-2(VNN2) protein as a novel biomarker highly-enriched in healthy subjects Mo-MDSC. Indeed, healthy subjects Mo-MDSC cells expressed 68 % VNN2, whereas only 9% VNN2 expression was observed on CD14+ HLA-DRhigh cells (n = 4 p < 0.01). The top 10 percent positive VNN2 monocytes expressed CD33 and CD11b while being negative for HLA-DR, CD3, CD15, CD19 and CD56, consistent with a Mo-MDSC phenotype. CD14+VNN2high monocytes were able to inhibit CD8 T cell proliferation comparably to traditional Mo-MDSC at 51 % and 48 % respectively. However, VNN2 expression on CD14+ monocytes from glioma patients was inversely correlated to their grade. CD14+VNN2high monocytes thus appear to mark a monocytic population similar to Mo-MDSC only in healthy subjects, which may be useful for tumor diagnoses.

Tumor-Infiltrating Myeloid Cells Co-Express TREM1 and TREM2 and Elevated TREM-1 Associates With Disease Progression in Renal Cell Carcinoma.

Myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) contribute to cancer-related inflammation and tumor progression. While several myeloid molecules have been ascribed a regulatory function in these processes, the triggering receptors expressed on myeloid cells (TREMs) have emerged as potent modulators of the innate immune response. While various TREMs amplify inflammation, others dampen it and are emerging as important players in modulating tumor progression-for instance, soluble TREM-1 (sTREM-1), which is detected during inflammation, associates with disease progression, while TREM-2 expression is associated with tumor-promoting macrophages. We hypothesized that TREM-1 and TREM-2 might be co-expressed on tumor-infiltrating myeloid cells and that elevated sTREM-1 associates with disease outcomes, thus representing a possibility for mutual modulation in cancer. Using the 4T1 breast cancer model, we found TREM-1 and TREM-2 expression on MDSC and TAM and that sTREM-1 was elevated in tumor-bearing mice in multiple models and correlated with tumor volume. While TREM-1 engagement enhanced TNF, a TREM-2 ligand was detected on MDSC and TAM, suggesting that both TREM could be functional in the tumor setting. Similarly, we detected TREM-1 and Trem2 expression in myeloid cells in the RENCA model of renal cell carcinoma (RCC). We confirmed these findings in human disease by demonstrating the expression of TREM-1 on tumor-infiltrating myeloid cells from patients with RCC and finding that sTREM-1 was increased in patients with RCC. Finally, The Cancer Genome Atlas analysis shows that TREM1 expression in tumors correlates with poor outcomes in RCC. Taken together, our data suggest that manipulation of the TREM-1/TREM-2 balance in tumors may be a novel means to modulate tumor-infiltrating myeloid cell phenotype and function.

The Bcl-2 transgene protects T cells from renal cell carcinoma-mediated apoptosis.

PURPOSE: Tumors induce T-cell apoptosis as a mechanism of inhibiting antitumor immunity. Using coculture experiments, it has been shown that tumor lines stimulate T-cell apoptosis by a pathway involving a mitochondrial permeability transition and cytochrome c release. Activated T cells express abundant levels of Bcl-2, an antiapoptotic molecule that would be expected to confer resistance to such tumor-mediated killing. We examined the mechanism by which Bcl-2 is dysregulated in T cells exposed to the renal tumor line SK-RC-45, and we determined whether overexpressing Bcl-2 protects T cells from tumor-mediated apoptosis. EXPERIMENTAL DESIGN: Activated T lymphocytes and Jurkat cells transfected or not transfected with Bcl-2 were exposed to SK-RC-45 for 48-72 h. After coculture, lymphocytes were analyzed for Bcl-2 expression using Western analysis and for tumor-induced apoptosis by terminal deoxynucleotidyl transferase-mediated nick end labeling. The role of SK-RC-45-stimulated caspase activation in degrading T-cell Bcl-2 was assessed using a pan-caspase inhibitor, as well as a specific inhibitor of caspase-9. RESULTS: The renal cell carcinoma cell line SK-RC-45 sensitizes peripheral blood activated T lymphocytes and Jurkat cells to apoptosis by a mechanism that involves degradation of the antiapoptotic protein Bcl-2. The SK-RC-45-induced modulation of lymphocyte Bcl-2 levels was largely caspase independent because pretreatment of T cells with pan-caspase inhibitor III or an inhibitor of caspase-9 had minimal or no effect on stabilizing the protein, although it did provide protection against apoptosis. Overexpression of Bcl-2 protected Jurkat cells from tumor-mediated killing. CONCLUSIONS: Bcl-2 inhibition is a mechanism by which tumors may render lymphocytes sensitive to other tumor-derived, proapoptotic stimuli.

Modification of the tumor microenvironment as a novel target of renal cell carcinoma therapeutics.

To move forward with immunotherapy, it is important to understand how the tumor microenvironment generates systemic immunosuppression in patients with renal cell carcinoma (RCC) as well as in patients with other types of solid tumors. Even though antigen discovery in RCC has lagged behind melanoma, recent clinical trials have finally authenticated that RCC is susceptible to vaccine-based therapy. Furthermore, judicious coadministration of cytokines and chemotherapy can potentiate therapeutic responses to vaccine in RCC and prolong survival, as has already proved possible for melanoma. Although high-dose interleukin 2 immunotherapy has been superseded as first-line therapy for RCC by promiscuous receptor tyrosine kinase inhibitors (rTKIs) such as sunitinib, sunitinib itself is a potent immunoadjunct in animal tumor models. A reasonable therapeutic goal is to unite antiangiogenic strategies with immunotherapy as first-line therapy for RCC. This strategy is equally appropriate for testing in all solid tumors in which the microenvironment generates immunosuppression. A common element of RCC and pancreatic, colon, breast, and other solid tumors is large numbers of circulating myeloid-derived suppressor cells (MDSCs), and because MDSCs elicit regulatory T cells rather than vice versa, gaining control over MDSCs is an important initial step in any immunotherapy. Although rTKIs like sunitinib have a remarkable capacity to deplete MDSCs and restore normal T-cell function in peripheral body compartments such as the bloodstream and the spleen, such rTKIs are effective only against MDSCs, which are engaged in phospho-STAT3-dependent programming (pSTAT3+). Unfortunately, rTKI-resistant pSTAT3- MDSCs are especially apt to arise within the tumor microenvironment itself, necessitating strategies that do not rely exclusively on STAT3 disruption. The most utilitarian strategy to gain control of both pSTAT3+ and pSTAT3- MDSCs may be to exploit the natural differentiation pathway, which permits MDSCs to mature into tumoricidal macrophages (TM1) via such stimuli as Toll-like receptor agonists, interferon γ, and CD40 ligation. Overall, this review highlights the mechanisms of immune suppression used by the different regulatory cell types operative in RCC as well as other tumors. It also describes the different therapeutic strategies to overcome the suppressive nature of the tumor microenvironment.

MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy.

Sunitinib is a receptor tyrosine kinase inhibitor (TKI) that is front-line therapy for metastatic renal cell carcinoma (mRCC). Its antitumor activity is related to its ability to block tumor cell and tumor vasculature cell signaling via several TKI receptors (i.e. vascular endothelial growth factor receptors VEGFRs, platelet-derived growth factors (PDGFs), and stem cell factors). Sunitinib also targets myeloid derived suppressor cells (MDSCs) significantly reducing their accumulation in the peripheral blood and reversing T cell (IFNγ) suppression in both mRCC patients and in murine tumor models. This reduction in immune suppression provides a rationale for combining sunitinib with immunotherapy for the treatment of certain tumor types. Despite these encouraging findings, however, we have observed that sunitinib has variable impact at reducing MDSCs and restoring T cell function within the tumor microenvironment. Given the immunosuppressive and proangiogenic activities of MDSC, it seems plausible that their persistence may contribute to the resistance that develops in sunitinib-treated patients. While sunitinib reduced tumor infiltrating MDSCs in Renca and CT26-bearing mice, coinciding with strong to modest decreases in tumor size respectively, it was ineffective at reducing MDSCs (<35% reduction in Gr1+CD11b+) or tumor burden in 4T1-bearing mice. Persistence of intratumor MDSCs was paralleled by depressed intratumor T cell IFNγ response and increased GM-CSF expression. Additionally, in vitro and in vivo experiments showed that GM-CSF prolongs survival of MDSCs, thus protecting them from the effects of sunitinib via a pSTAT5-dependent pathway. Although preliminary, there is evidence of intratumor MDSC resistance in some mRCC patients following sunitinib treatment. Intratumor MDSC persistence and T cell IFNγ response post nephrectomy in patients receiving sunitinib in a neoadjuvant setting are being compared to RCC patients undergoing nephrectomy without prior sunitinib treatment. Tumors from untreated patients showed suppressed T cell IFNγ response along with substantial expression of MDSCs (5% of total digested cells). Thus far, tumors from 5/8 neoadjuvant patients showed persistence of intratumor MDSCs and low T cell IFNγ production post sunitinib treatment, findings that parallel results from untreated tumors. In the remaining 3 neoadjuvant patients, intratumor MDSCs were detected at low levels which coincided with a T cell IFNγ response similar to that observed with normal donor peripheral T cells. GM-CSF's role in promoting MDSC survival in patient tumors is supported by the observation that GM-CSF is produced in short-term RCC cultures at levels capable of protecting MDSCs from sunitinib-induced cell death. Additionally, persistence of MDSC also may be associated with increased expression of proangiogenic proteins, such as MMP9, MMP8, and IL-8 produced by tumor stromal cells or infiltrating MDSCs. Indeed our findings suggest that the most dominate MDSC subset in RCC patients is the neutrophilic population that produces proangiogenic proteins. We propose that the development of sunitinib resistance is partly mediated by the survival of MDSCs intratumorally, thereby providing sustained immune suppression and angiogenesis.