In situ thermal ablation augments antitumor efficacy of adoptive T cell therapy.

Purpose: The aim of this study is to investigate whether radiofrequency ablation (RFA) improves the efficacy of adoptive T cell immunotherapy in preclinical mouse cancer models.Method: Mice implanted subcutaneously (sc) with syngeneic colon adenocarcinoma or melanoma were treated with sub-curative in situ RFA (90 °C, 1 min). Trafficking of T cells to lymph nodes (LN) or tumors was quantified by homing assays and intravital microscopy (IVM) after sham procedure or RFA. Expression of trafficking molecules (CCL21 and intercellular adhesion molecule-1 [ICAM-1]) on high endothelial venules (HEV) in LN and tumor vessels was evaluated by immunofluorescence microscopy. Tumor-bearing mice were pretreated with RFA to investigate the therapeutic benefit when combined with adoptive transfer of in vitro-activated tumor-specific CD8+ T cells.Results: RFA increased trafficking of naïve CD8+ T cells to tumor-draining LN (TdLN). A corresponding increase in expression of ICAM-1 and CCL21 was detected on HEV in TdLN but not in contralateral (c)LN. IVM revealed that RFA substantially enhanced secondary firm arrest of lymphocytes selectively in HEV in TdLN. Furthermore, strong induction of ICAM-1 in tumor vessels was associated with significantly augmented trafficking of adoptively transferred in vitro-activated CD8+ T cells to tumors after RFA. Finally, preconditioning tumors with RFA augmented CD8+ T cell-mediated apoptosis of tumor targets and delayed growth of established tumors when combined with adoptive T cell transfer immunotherapy.Conclusions: These studies suggest that in addition to its role as a palliative therapeutic modality, RFA may have clinical potential as an immune-adjuvant therapy by augmenting the efficacy of adoptive T cell therapy.

Dual inhibition of Ang-2 and VEGF receptors normalizes tumor vasculature and prolongs survival in glioblastoma by altering macrophages.

Glioblastomas (GBMs) rapidly become refractory to anti-VEGF therapies. We previously demonstrated that ectopic overexpression of angiopoietin-2 (Ang-2) compromises the benefits of anti-VEGF receptor (VEGFR) treatment in murine GBM models and that circulating Ang-2 levels in GBM patients rebound after an initial decrease following cediranib (a pan-VEGFR tyrosine kinase inhibitor) administration. Here we tested whether dual inhibition of VEGFR/Ang-2 could improve survival in two orthotopic models of GBM, Gl261 and U87. Dual therapy using cediranib and MEDI3617 (an anti-Ang-2-neutralizing antibody) improved survival over each therapy alone by delaying Gl261 growth and increasing U87 necrosis, effectively reducing viable tumor burden. Consistent with their vascular-modulating function, the dual therapies enhanced morphological normalization of vessels. Dual therapy also led to changes in tumor-associated macrophages (TAMs). Inhibition of TAM recruitment using an anti-colony-stimulating factor-1 antibody compromised the survival benefit of dual therapy. Thus, dual inhibition of VEGFR/Ang-2 prolongs survival in preclinical GBM models by reducing tumor burden, improving normalization, and altering TAMs. This approach may represent a potential therapeutic strategy to overcome the limitations of anti-VEGFR monotherapy in GBM patients by integrating the complementary effects of anti-Ang2 treatment on vessels and immune cells.

Generation of functionally competent and durable engineered blood vessels from human induced pluripotent stem cells.

Efficient generation of competent vasculogenic cells is a critical challenge of human induced pluripotent stem (hiPS) cell-based regenerative medicine. Biologically relevant systems to assess functionality of the engineered vessels in vivo are equally important for such development. Here, we report a unique approach for the derivation of endothelial precursor cells from hiPS cells using a triple combination of selection markers--CD34, neuropilin 1, and human kinase insert domain-containing receptor--and an efficient 2D culture system for hiPS cell-derived endothelial precursor cell expansion. With these methods, we successfully generated endothelial cells (ECs) from hiPS cells obtained from healthy donors and formed stable functional blood vessels in vivo, lasting for 280 d in mice. In addition, we developed an approach to generate mesenchymal precursor cells (MPCs) from hiPS cells in parallel. Moreover, we successfully generated functional blood vessels in vivo using these ECs and MPCs derived from the same hiPS cell line. These data provide proof of the principle that autologous hiPS cell-derived vascular precursors can be used for in vivo applications, once safety and immunological issues of hiPS-based cellular therapy have been resolved. Additionally, the durability of hiPS-derived blood vessels in vivo demonstrates a potential translation of this approach in long-term vascularization for tissue engineering and treatment of vascular diseases. Of note, we have also successfully generated ECs and MPCs from type 1 diabetic patient-derived hiPS cell lines and use them to generate blood vessels in vivo, which is an important milestone toward clinical translation of this approach.

Phase I and Biomarker Study of Plerixafor and Bevacizumab in Recurrent High-Grade Glioma.

Purpose: Although antiangiogenic therapy for high-grade glioma (HGG) is promising, responses are not durable. Correlative clinical studies suggest that the SDF-1α/CXCR4 axis may mediate resistance to VEGFR inhibition. Preclinical data have demonstrated that plerixafor (a reversible CXCR4 inhibitor) could inhibit glioma progression after anti-VEGF pathway inhibition. We conducted a phase I study to determine the safety of plerixafor and bevacizumab in recurrent HGG.Patients and Methods: Part 1 enrolled 23 patients with a 3 × 3 dose escalation design to a maximum planned dose of plerixafor 320 µg/kg subcutaneously on days 1 to 21 and bevacizumab 10 mg/kg intravenously on days 1 and 15 of each 28-day cycle. Cerebrospinal fluid (CSF) and plasma samples were obtained for pharmacokinetic analyses. Plasma and cellular biomarkers were evaluated before and after treatment. Part 2 enrolled 3 patients and was a surgical study to determine plerixafor's penetration in tumor tissue.Results: In Part 1, no dose-limiting toxicities were seen at the maximum planned dose of plerixafor + bevacizumab. Treatment was well tolerated. After plerixafor 320 µg/kg treatment, the average CSF drug concentration was 26.8 ± 19.6 ng/mL. Plerixafor concentration in resected tumor tissue from patients pretreated with plerixafor was 10 to 12 µg/g. Circulating biomarker data indicated that plerixafor + bevacizumab induces rapid and persistent increases in plasma SDF-1α and placental growth factor. Progression-free survival correlated with pretreatment plasma soluble mesenchymal-epithelial transition receptor and sVEGFR1, and overall survival with the change during treatment in CD34+ progenitor/stem cells and CD8 T cells.Conclusions: Plerixafor + bevacizumab was well tolerated in HGG patients. Plerixafor distributed to both the CSF and brain tumor tissue, and treatment was associated with biomarker changes consistent with VEGF and CXCR4 inhibition. Clin Cancer Res; 24(19); 4643-9. ©2018 AACR.

Obesity promotes resistance to anti-VEGF therapy in breast cancer by up-regulating IL-6 and potentially FGF-2.

Anti-vascular endothelial growth factor (VEGF) therapy has failed to improve survival in patients with breast cancer (BC). Potential mechanisms of resistance to anti-VEGF therapy include the up-regulation of alternative angiogenic and proinflammatory factors. Obesity is associated with hypoxic adipose tissues, including those in the breast, resulting in increased production of some of the aforementioned factors. Hence, we hypothesized that obesity could contribute to anti-VEGF therapy's lack of efficacy. We found that BC patients with obesity harbored increased systemic concentrations of interleukin-6 (IL-6) and/or fibroblast growth factor 2 (FGF-2), and their tumor vasculature was less sensitive to anti-VEGF treatment. Mouse models revealed that obesity impairs the effects of anti-VEGF on angiogenesis, tumor growth, and metastasis. In one murine BC model, obesity was associated with increased IL-6 production from adipocytes and myeloid cells within tumors. IL-6 blockade abrogated the obesity-induced resistance to anti-VEGF therapy in primary and metastatic sites by directly affecting tumor cell proliferation, normalizing tumor vasculature, alleviating hypoxia, and reducing immunosuppression. Similarly, in a second mouse model, where obesity was associated with increased FGF-2, normalization of FGF-2 expression by metformin or specific FGF receptor inhibition decreased vessel density and restored tumor sensitivity to anti-VEGF therapy in obese mice. Collectively, our data indicate that obesity fuels BC resistance to anti-VEGF therapy via the production of inflammatory and angiogenic factors.

Langerhans Cells Orchestrate TFH-Dependent Humoral Immunity.

T follicular helper cells contribute to the development of long-lasting humoral immunity by germinal center formation. Somatic hypermutation and affinity maturation take place in germinal centers leading to the generation of memory B cells and plasma cells. As such, T follicular helper cells impact immunodeficiencies, autoimmunity, and cancer. This necessitates further understanding of how T follicular helper cells are regulated in health and disease. The current study by Levin et al. builds on prior work to further substantiate a critical role for skin migratory dendritic cells and in particular Langerhans cells at governing T follicular helper and germinal center formation after intradermal immunization with HIV p24-coated polylactic acid nanoparticles.

Preclinical Efficacy of Ado-trastuzumab Emtansine in the Brain Microenvironment.

BACKGROUND: Central nervous system (CNS) metastases represent a major problem in the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer because of the disappointing efficacy of HER2-targeted therapies against brain lesions. The antibody-drug conjugate ado-trastuzumab emtansine (T-DM1) has shown efficacy in trastuzumab-resistant systemic breast cancer. Here, we tested the hypothesis that T-DM1 could overcome trastuzumab resistance in murine models of brain metastases. METHODS: We treated female nude mice bearing BT474 or MDA-MB-361 brain metastases (n = 9-11 per group) or cancer cells grown in organotypic brain slice cultures with trastuzumab or T-DM1 at equivalent or equipotent doses. Using intravital imaging, molecular techniques and histological analysis we determined tumor growth, mouse survival, cancer cell apoptosis and proliferation, tumor drug distribution, and HER2 signaling. Data were analyzed with one-way analysis of variance (ANOVA), Kaplan-Meier analysis, and Coefficient of Determination. All statistical tests were two-sided. RESULTS: T-DM1 delayed the growth of HER2-positive breast cancer brain metastases compared with trastuzumab. These findings were consistent between HER2-driven and PI3K-driven tumors. The activity of T-DM1 resulted in a survival benefit (median survival for BT474 tumors: 28 days for trastuzumab vs 112 days for T-DM1, hazard ratio = 6.2, 95% confidence interval = 6.1 to 85.84, P < .001). No difference in drug distribution or HER2-signaling was revealed between the two groups. However, T-DM1 led to a statistically significant increase in tumor cell apoptosis (one-way ANOVA for ApopTag, P < .001), which was associated with mitotic catastrophe. CONCLUSIONS: T-DM1 can overcome resistance to trastuzumab therapy in HER2-driven or PI3K-driven breast cancer brain lesions due to the cytotoxicity of the DM1 component. Clinical investigation of T-DM1 for patients with CNS metastases from HER2-positive breast cancer is warranted.

PlGF/VEGFR-1 Signaling Promotes Macrophage Polarization and Accelerated Tumor Progression in Obesity.

PURPOSE: Obesity promotes pancreatic and breast cancer progression via mechanisms that are poorly understood. Although obesity is associated with increased systemic levels of placental growth factor (PlGF), the role of PlGF in obesity-induced tumor progression is not known. PlGF and its receptor VEGFR-1 have been shown to modulate tumor angiogenesis and promote tumor-associated macrophage (TAM) recruitment and activity. Here, we hypothesized that increased activity of PlGF/VEGFR-1 signaling mediates obesity-induced tumor progression by augmenting tumor angiogenesis and TAM recruitment/activity. EXPERIMENTAL DESIGN: We established diet-induced obese mouse models of wild-type C57BL/6, VEGFR-1 tyrosine kinase (TK)-null, or PlGF-null mice, and evaluated the role of PlGF/VEGFR-1 signaling in pancreatic and breast cancer mouse models and in human samples. RESULTS: We found that obesity increased TAM infiltration, tumor growth, and metastasis in pancreatic cancers, without affecting vessel density. Ablation of VEGFR-1 signaling prevented obesity-induced tumor progression and shifted the tumor immune environment toward an antitumor phenotype. Similar findings were observed in a breast cancer model. Obesity was associated with increased systemic PlGF, but not VEGF-A or VEGF-B, in pancreatic and breast cancer patients and in various mouse models of these cancers. Ablation of PlGF phenocopied the effects of VEGFR-1-TK deletion on tumors in obese mice. PlGF/VEGFR-1-TK deletion prevented weight gain in mice fed a high-fat diet, but exacerbated hyperinsulinemia. Addition of metformin not only normalized insulin levels but also enhanced antitumor immunity. CONCLUSIONS: Targeting PlGF/VEGFR-1 signaling reprograms the tumor immune microenvironment and inhibits obesity-induced acceleration of tumor progression. Clin Cancer Res; 22(12); 2993-3004. ©2016 AACR.

Targeting the Tumor Microenvironment to Enhance Pediatric Brain Cancer Treatment.

Strategies targeting the microenvironment of pediatric brain cancers have the potential to improve the efficacy of standard and genome-based molecular therapeutics. These strategies also have the potential of helping resolve many of the challenges associated with developing new drugs and running clinical trials for relatively small pediatric brain tumor population. Disrupting vital paracrine and physical interactions between cancer cells and surrounding stroma, targeting and normalizing the abnormal tumor vasculature, and/or inducing antitumor immunity represent some of the most promising approaches. A comprehensive characterization of the pediatric brain tumor microenvironment's composition and function and its modulation by chemoradiation and molecularly targeted therapies is warranted to develop and effectively implement these approaches.

IL-6 trans-signaling licenses mouse and human tumor microvascular gateways for trafficking of cytotoxic T cells.

Immune cells are key regulators of neoplastic progression, which is often mediated through their release of cytokines. Inflammatory cytokines such as IL-6 exert tumor-promoting activities by driving growth and survival of neoplastic cells. However, whether these cytokines also have a role in recruiting mediators of adaptive anticancer immunity has not been investigated. Here, we report that homeostatic trafficking of tumor-reactive CD8+ T cells across microvascular checkpoints is limited in tumors despite the presence of inflammatory cytokines. Intravital imaging in tumor-bearing mice revealed that systemic thermal therapy (core temperature elevated to 39.5°C ± 0.5°C for 6 hours) activated an IL-6 trans-signaling program in the tumor blood vessels that modified the vasculature such that it could support enhanced trafficking of CD8+ effector/memory T cells (Tems) into tumors. A concomitant decrease in tumor infiltration by Tregs during systemic thermal therapy resulted in substantial enhancement of Tem/Treg ratios. Mechanistically, IL-6 produced by nonhematopoietic stromal cells acted cooperatively with soluble IL-6 receptor-α and thermally induced gp130 to promote E/P-selectin- and ICAM-1-dependent extravasation of cytotoxic T cells in tumors. Parallel increases in vascular adhesion were induced by IL-6/soluble IL-6 receptor-α fusion protein in mouse tumors and patient tumor explants. Finally, a causal link was established between IL-6-dependent licensing of tumor vessels for Tem trafficking and apoptosis of tumor targets. These findings suggest that the unique IL-6-rich tumor microenvironment can be exploited to create a therapeutic window to boost T cell-mediated antitumor immunity and immunotherapy.

Fine-tuning immune surveillance by fever-range thermal stress.

An effectively orchestrated immune response to infection and disease depends on efficient trafficking of lymphocytes across vascular beds at distinct tissue sites. Local inflammation and systemic fever increase immune surveillance to immune-relevant sites throughout the body. During the initiation phase of inflammation, this tightly regulated process improves leukocyte trafficking to the secondary lymphoid organs where they undergo activation and expansion in response to cognate antigen. In the resolution phase following the clearance of the invading pathogen, lymphocyte entry is rapidly returned to baseline conditions. Specialized blood vessels termed high endothelial venules (HEVs) have emerged as critical 'hotspots' controlling the rate of lymphocyte entry into lymphoid organs during both phases of inflammation. In this review, we will examine the remarkably tight regulation of lymphocyte trafficking across HEVs conferred by inflammatory cues associated with the thermal element of fever. These studies have revealed a novel role for interleukin-6 (IL-6) trans-signaling in eliciting systemic effects on lymphocyte trafficking patterns to fine-tune immune surveillance.

Regulation of a lymphocyte-endothelial-IL-6 trans-signaling axis by fever-range thermal stress: hot spot of immune surveillance.

The pleiotropic cytokine, interleukin-6 (IL-6), has emerged in recent years as a key regulator of the transition from innate to adaptive immunity through its ability to modulate leukocyte recruitment at inflammatory sites. This review highlights a newly identified role for IL-6 trans-signaling, initiated by an agonistic complex of IL-6 and a soluble form of IL-6 receptor alpha, in heightening immune surveillance of peripheral lymphoid organs during febrile inflammatory responses. Inflammatory cues provided by the thermal component of fever trigger IL-6 trans-signaling to act at discrete levels in the multistep adhesion cascade that governs the entry of blood-borne lymphocytes across 'gatekeeper' high endothelial venules (HEVs) in lymph nodes and Peyer patches. IL-6 trans-signaling-dependent mechanisms have been elucidated during thermal stimulation of primary tethering and rolling of lymphocytes along the lumenal surface of HEVs as well as during secondary firm arrest of lymphocytes in HEVs prior to their migration into the underlying parenchyma. These mechanisms profoundly increase the probability that lymphocytes that continuously patrol the body will engage in productive encounters with target antigens sequestered within lymphoid organs. Findings that the lymphocyte-HEV-IL-6 trans-signaling biological axis functions as a thermally-sensitive alert system that promotes immune surveillance provide insight into one of the unresolved mysteries in immunology regarding the benefits of mounting a febrile reaction during inflammation.