A rationally designed combined treatment with an alphavirus-based cancer vaccine, sunitinib and low-dose tumor irradiation completely blocks tumor development.

The clinical efficacy of therapeutic cancer vaccines remains limited. For effective immunotherapeutic responses in cancer patients, multimodal approaches capable of both inducing antitumor immune responses and bypassing tumor-mediated immune escape seem essential. Here, we report on a combination therapy comprising sunitinib (40 mg/kg), single low-dose (14 Gy) tumor irradiation and immunization with a therapeutic cancer vaccine based on a Semliki Forest virus vector encoding the oncoproteins E6 and E7 of human papillomavirus (SFVeE6,7). We previously demonstrated that either low-dose irradiation or sunitinib in single combination with SFVeE6,7 immunizations enhanced the intratumoral ratio of antitumor effector cells to myeloid-derived suppressor cells (MDSCs). On the basis of these results we designed a triple treatment combinatorial regimen. The trimodal sunitinib, low-dose irradiation and SFVeE6,7 immunization therapy resulted in stronger intratumoral MDSC depletion than sunitinib alone. Concomitantly, the highest levels of intratumoral E7-specific CD8+ T cells were attained after triple treatment. Approximately 75% of these cells were positive for the early activation marker CD69. The combination of sunitinib, low-dose tumor irradiation and SFVeE6,7 immunization dramatically changed the intratumoral immune compartment. Whereas control tumors contained 0.02 E7-specific CD8+ T cells per MDSC, triple treatment tumors contained more than 200 E7-specific CD8+ T cells per MDSC, a 10,000-fold increased ratio. As a result, the triple treatment strongly enhanced the immunotherapeutic antitumor effect, blocking tumor development altogether and leading to 100% tumor-free survival of tumor-bearing mice. This study demonstrates that this multimodal approach elicits superior antitumor effects and should be considered for clinical applications.

Myeloid derived suppressor cells-An overview of combat strategies to increase immunotherapy efficacy.

Myeloid-derived suppressor cells (MDSCs) contribute to tumor-mediated immune escape and negatively correlate with overall survival of cancer patients. Nowadays, a variety of methods to target MDSCs are being investigated. Based on the intervention stage of MDSCs, namely development, expansion and activation, function and turnover, these methods can be divided into: (I) prevention or differentiation to mature cells, (II) blockade of MDSC expansion and activation, (III) inhibition of MDSC suppressive activity or (IV) depletion of intratumoral MDSCs. This review describes effective mono- or multimodal-therapies that target MDSCs for the benefit of cancer treatment.

Sunitinib depletes myeloid-derived suppressor cells and synergizes with a cancer vaccine to enhance antigen-specific immune responses and tumor eradication.

The high efficacy of therapeutic cancer vaccines in preclinical studies has yet to be fully achieved in clinical trials. Tumor immune suppression is a critical factor that hampers the desired antitumor effect. Here, we analyzed the combined effect of a cancer vaccine and the receptor tyrosine kinase inhibitor sunitinib. Sunitinib was administered intraperitoneally, alone or in combination with intramuscular immunization using a viral vector based cancer vaccine composed of Semliki Forest virus replicon particles and encoding the oncoproteins E6 and E7 (SFVeE6,7) of human papilloma virus (HPV). We first demonstrated that treatment of tumor-bearing mice with sunitinib alone dose-dependently depleted myeloid-derived suppressor cells (MDSCs) in the tumor, spleen and in circulation. Concomitantly, the number of CD8+ T cells increased 2-fold and, on the basis of CD69 expression, their activation status was greatly enhanced. The intrinsic immunosuppressive activity of residual MDSCs after sunitinib treatment was not changed in a dose-dependent fashion. We next combined sunitinib treatment with SFVeE6,7 immunization. This combined treatment resulted in a 1.5- and 3-fold increase of E7-specific cytotoxic T lymphocytes (CTLs) present within the circulation and tumor, respectively, as compared to immunization only. The ratio of E7-specific CTLs to MDSCs in blood thereby increased 10- to 20-fold and in tumors up to 12.5-fold. As a result, the combined treatment strongly enhanced the antitumor effect of the cancer vaccine. This study demonstrates that sunitinib creates a favorable microenvironment depleted of MDSCs and acts synergistically with a cancer vaccine resulting in enhanced levels of active tumor-antigen specific CTLs, thus changing the balance in favor of antitumor immunity.

Therapeutic immunization and local low-dose tumor irradiation, a reinforcing combination.

Therapeutic cancer vaccines show promise in preclinical studies, yet their clinical efficacy is limited. Increased recruitment of immune cells into tumors and suppression of the immune suppressive tumor environment are critical components toward effective cancer immunotherapies. Here, we report how local low-dose irradiation, alone or with a therapeutic immunization based on Semliki Forest virus (SFV) against human papillomavirus (HPV)-related cancer, influences these immune mechanisms. We first demonstrated that immunization with SFVeE6,7 or SFVeOVA, replicon particles expressing either HPV16 E6/E7 or ovalbumin, resulted in an antigen-specific migration of CD8+ T cells into HPV- and OVA-specific tumors. Local low-dose tumor irradiation alone resulted in a 2-fold increase of intratumoral CD8+ T cells. When 14 Gy irradiation was combined with immunization, intratumoral numbers of CD8+ T cells increased 10-fold and the number of CD8+ T cells specific for the E7- epitope increased more than 20-fold. Irradiation alone however also increased the number of intratumoral myeloid-derived suppressor cells (MDSCs) 3.5-fold. Importantly, this number did not further increase when combined with immunization. As a result, the ratio of antigen-specific CD8+ T cells and MDSCs in tumors increased up to 85-fold compared to the control. We furthermore demonstrated that following irradiation CCR2 and CCL2, CXCR6 and CCL16, chemokines and ligands involved in tumor homing of immune cells, were significantly up regulated. This study demonstrates that local low-dose tumor irradiation influences the intratumoral immune population induced by SFVeE6,7 immunization by a strong increase in the ratio of antitumoral to immune suppressive cells, thus changing the intratumoral immune balance in favor of antitumor activity.

The function of the ATP-binding cassette (ABC) transporter ABCB1 is not susceptible to actin disruption.

Previously we have shown that the activity of the multidrug transporter ABCC1 (multidrug resistance protein 1), and its localization in lipid rafts, depends on cortical actin (Hummel I, Klappe K, Ercan C, Kok JW. Mol. Pharm. 2011 79, 229-40). Here we show that the efflux activity of the ATP-binding cassette (ABC) family member ABCB1 (P-glycoprotein), did not depend on actin, neither in ABCB1 over expressing murine National Institutes of Health (NIH) 3T3 MDR1 G185 cells nor in human SK-N-FI cells, which endogenously express ABCB1. Disruption of the actin cytoskeleton, upon treatment of the cells with latrunculin B or cytochalasin D, caused severe changes in cell and membrane morphology, and concomitant changes in the subcellular distribution of ABCB1, as revealed by confocal laser scanning and electron microscopy. Nevertheless, irrespective of actin perturbation, the cell surface pool of ABCB1 remained unaltered. In NIH 3T3 MDR1 G185 cells, ABCB1 is partly localized in detergent-free lipid rafts, which partitioned in two different density gradient regions, both enriched in cholesterol and sphingolipids. Interestingly, disruption of the actin cytoskeleton did not change the density gradient distribution of ABCB1. Our data demonstrate that the functioning of ABCB1 as an efflux pump does not depend on actin, which is due to its distribution in both cell surface-localized non-raft membrane areas and lipid raft domains, which do not depend on actin stabilization.

From tumor immunosuppression to eradication: targeting homing and activity of immune effector cells to tumors.

Unraveling the mechanisms used by the immune system to fight cancer development is one of the most ambitious undertakings in immunology. Detailed knowledge regarding the mechanisms of induction of tolerance and immunosuppression within the tumor microenvironment will contribute to the development of highly effective tumor eradication strategies. Research within the last few decades has shed more light on the matter. This paper aims to give an overview on the current knowledge of the main tolerance and immunosuppression mechanisms elicited within the tumor microenvironment, with the focus on development of effective immunotherapeutic strategies to improve homing and activity of immune effector cells to tumors.

Low body temperature governs the decline of circulating lymphocytes during hibernation through sphingosine-1-phosphate.

Hibernation is an energy-conserving behavior consisting of periods of inhibited metabolism ('torpor') with lowered body temperature. Torpor bouts are interspersed by arousal periods, in which metabolism increases and body temperature returns to euthermia. In deep torpor, the body temperature typically decreases to 2-10 °C, and major physiological and immunological changes occur. One of these alterations constitutes an almost complete depletion of circulating lymphocytes that is reversed rapidly upon arousal. Here we show that torpor induces the storage of lymphocytes in secondary lymphoid organs in response to a temperature-dependent drop in plasma levels of sphingosine-1-phosphate (S1P). Regulation of lymphocyte numbers was mediated through the type 1 S1P receptor (S1P(1)), because administration of a specific antagonist (W146) during torpor (in a Syrian hamster at ∼8 °C) precluded restoration of lymphocyte numbers upon subsequent arousal. Furthermore, S1P release from erythrocytes via ATP-binding cassette (ABC)-transporters was significantly inhibited at low body temperature (4 °C) but was restored upon rewarming. Reversible lymphopenia also was observed during daily torpor (in a Djungarian hamster at ± 25 °C), during forced hypothermia in anesthetized (summer-active) hamsters (at ± 9 °C), and in a nonhibernator (rat at ∼19 °C). Our results demonstrate that lymphopenia during hibernation in small mammals is driven by body temperature, via altered plasma S1P levels. S1P is recognized as an important bioactive lipid involved in regulating several other physiological processes as well and may be an important factor regulating additional physiological processes in hibernation as well as in mediating the effects of therapeutic hypothermia in patients.