Inflammation- and tumor-induced anorexia and weight loss require MyD88 in hematopoietic/myeloid cells but not in brain endothelial or neural cells.

Loss of appetite is a hallmark of inflammatory diseases. The underlying mechanisms remain undefined, but it is known that myeloid differentiation primary response gene 88 (MyD88), an adaptor protein critical for Toll-like and IL-1 receptor family signaling, is involved. Here we addressed the question of determining in which cells the MyD88 signaling that results in anorexia development occurs by using chimeric mice and animals with cell-specific deletions. We found that MyD88-knockout mice, which are resistant to bacterial lipopolysaccharide (LPS)-induced anorexia, displayed anorexia when transplanted with wild-type bone marrow cells. Furthermore, mice with a targeted deletion of MyD88 in hematopoietic or myeloid cells were largely protected against LPS-induced anorexia and displayed attenuated weight loss, whereas mice with MyD88 deletion in hepatocytes or in neural cells or the cerebrovascular endothelium developed anorexia and weight loss of similar magnitude as wild-type mice. Furthermore, in a model for cancer-induced anorexia-cachexia, deletion of MyD88 in hematopoietic cells attenuated the anorexia and protected against body weight loss. These findings demonstrate that MyD88-dependent signaling within the brain is not required for eliciting inflammation-induced anorexia. Instead, we identify MyD88 signaling in hematopoietic/myeloid cells as a critical component for acute inflammatory-driven anorexia, as well as for chronic anorexia and weight loss associated with malignant disease.

Hiwi mediated tumorigenesis is associated with DNA hypermethylation.

Expression of Piwi proteins is confined to early development and stem cells during which they suppress transposon migration via DNA methylation to ensure genomic stability. Piwi's genomic protective function conflicts with reports that its human ortholog, Hiwi, is expressed in numerous cancers and prognosticates shorter survival. However, the role of Hiwi in tumorigenesis has not been examined. Here we demonstrate that (1) over-expressing Hiwi in sarcoma precursors inhibits their differentiation in vitro and generates sarcomas in vivo; (2) transgenic mice expressing Hiwi (mesodermally restricted) develop sarcomas; and (3) inducible down-regulation of Hiwi in human sarcomas inhibits growth and re-establishes differentiation. Our data indicates that Hiwi is directly tumorigenic and Hiwi-expressing cancers may be addicted to Hiwi expression. We further show that Hiwi associated DNA methylation and cyclin-dependent kinase inhibitor (CDKI) silencing is reversible along with Hiwi-induced tumorigenesis, via DNA-methyltransferase inhibitors. Our studies reveal for the first time not only a novel oncogenic role for Hiwi as a driver of tumorigenesis, but also suggest that the use of epigenetic agents may be clinically beneficial for treatment of tumors that express Hiwi. Additionally, our data showing that Hiwi-associated DNA hyper-methylation with subsequent genetic and epigenetic changes favoring a tumorigenic state reconciles the conundrum of how Hiwi may act appropriately to promote genomic integrity during early development (via transposon silencing) and inappropriately in adult tissues with subsequent tumorigenesis.

Contribution of IL-17-producing gamma delta T cells to the efficacy of anticancer chemotherapy.

By triggering immunogenic cell death, some anticancer compounds, including anthracyclines and oxaliplatin, elicit tumor-specific, interferon-γ-producing CD8(+) αß T lymphocytes (Tc1 CTLs) that are pivotal for an optimal therapeutic outcome. Here, we demonstrate that chemotherapy induces a rapid and prominent invasion of interleukin (IL)-17-producing γδ (Vγ4(+) and Vγ6(+)) T lymphocytes (γδ T17 cells) that precedes the accumulation of Tc1 CTLs within the tumor bed. In T cell receptor δ(-/-) or Vγ4/6(-/-) mice, the therapeutic efficacy of chemotherapy was compromised, no IL-17 was produced by tumor-infiltrating T cells, and Tc1 CTLs failed to invade the tumor after treatment. Although γδ T17 cells could produce both IL-17A and IL-22, the absence of a functional IL-17A-IL-17R pathway significantly reduced tumor-specific T cell responses elicited by tumor cell death, and the efficacy of chemotherapy in four independent transplantable tumor models. Adoptive transfer of γδ T cells restored the efficacy of chemotherapy in IL-17A(-/-) hosts. The anticancer effect of infused γδ T cells was lost when they lacked either IL-1R1 or IL-17A. Conventional helper CD4(+) αß T cells failed to produce IL-17 after chemotherapy. We conclude that γδ T17 cells play a decisive role in chemotherapy-induced anticancer immune responses.

OX40-enhanced tumor rejection and effector T cell differentiation decreases with age.

OX40 agonists have potent immunotherapeutic effects against a variety of murine tumors, yet it is unclear the role that age-related immune senescence plays on their efficacy. We found that middle-aged and elderly tumor-bearing mice (12 and 20 mo old, respectively) treated with anti-OX40 were less responsive compared with young mice 6 mo or less of age. Decreased tumor-free survival was observed in both male and female mice, and was not due to changes in the surface expression of OX40 on T cells in older animals. Enumeration of cytokine-producing effector T cells in tumor-bearing mice revealed a significant decline in these cells in the older mice treated with anti-OX40 compared with their younger counterparts. The decrease of this critical T cell population in middle-aged mice was not a result of inherent T cell deficiencies, but was revealed to be T cell extrinsic. Finally, combining IL-12, an innate cytokine, with anti-OX40 boosted levels of differentiated effector T cells in the older anti-OX40-treated mice and partially restored the defective antitumor responses in the middle-aged mice. Our data show that the anti-OX40-enhancement of tumor immunity and effector T cell numbers is decreased in middle-aged mice and was partially reversed by coadministration of the proinflammatory cytokine IL-12.

[Influence of Laminaria japonica and fucus on hemostasis and sarcoma M-1 growth in Wistar rats].

The study was concerned with the effects of brown seaweeds on hemostasis and sarcoma M-1 growth in rats. Laminaria japonica and focus were shown to exert anticoagulant action to inhibit carcinogenesis. They may be used for adjuvant therapy of tumors.

Acute and chronic administration of the cannabinoid receptor agonist CP 55,940 attenuates tumor-evoked hyperalgesia.

Patients with cancer frequently report pain that can be difficult to manage. This study examined the antihyperalgesic effects of a cannabinoid receptor agonist, CP 55,940, in a murine model of cancer pain. Implantation of fibrosarcoma cells into and around the calcaneous bone in mice produced mechanical hyperalgesia (decreased paw withdrawal thresholds and increased frequency of paw withdrawals). On day 13 after implantation, mechanical hyperalgesia, nociception, and catalepsy were assessed. Mice were randomly assigned to receive CP 55,940 (0.01-3 mg/kg, i.p.) or vehicle and behavioral measures were redetermined. CP 55,940 dose-dependently attenuated tumor-evoked mechanical hyperalgesia. To examine the effect of catalepsy on the antihyperalgesic effect of CP 55,940, mice with tumor-evoked hyperalgesia were pretreated with the dopamine agonist apomorphine prior to administration of CP 55,940. Apomorphine attenuated the cataleptic effect of CP 55,940 but did not attenuate its antihyperalgesic effect. In a separate group of mice with tumor-evoked hyperalgesia, administration of the dopamine antagonist spiperone produced catalepsy that was approximately 2.5 fold greater than that produced by CP 55,490. Whereas this dose of CP 55,940 completely reversed tumor-evoked mechanical hyperalgesia, spiperone only attenuated mechanical hyperalgesia by approximately 35%. Thus, the cataleptic effects of CP 55,940 did not fully account for its antihyperalgesic effect. The antihyperalgesic effect of CP 55,940 was mediated via the cannabinoid CB1 but not CB2 receptor. Finally, repeated administration of CP 55,940 produced a short-term and a longer-term attenuation of tumor-evoked hyperalgesia. These results suggest that cannabinoids may be a useful alternative or adjunct therapy for treating cancer pain.

Noninvasive monitoring of murine tumor blood flow during and after photodynamic therapy provides early assessment of therapeutic efficacy.

PURPOSE: To monitor tumor blood flow noninvasively during photodynamic therapy (PDT) and to correlate flow responses with therapeutic efficacy. EXPERIMENTAL DESIGN: Diffuse correlation spectroscopy (DCS) was used to measure blood flow continuously in radiation-induced fibrosarcoma murine tumors during Photofrin (5 mg/kg)/PDT (75 mW/cm2, 135 J/cm2). Relative blood flow (rBF; i.e., normalized to preillumination values) was compared with tumor perfusion as determined by power Doppler ultrasound and was correlated with treatment durability, defined as the time of tumor growth to a volume of 400 mm3. Broadband diffuse reflectance spectroscopy concurrently quantified tumor hemoglobin oxygen saturation (SO2). RESULTS: DCS and power Doppler ultrasound measured similar flow decreases in animals treated with identical protocols. DCS measurement of rBF during PDT revealed a series of PDT-induced peaks and declines dominated by an initial steep increase (average +/- SE: 168.1 +/- 39.5%) and subsequent decrease (59.2 +/- 29.1%). The duration (interval time; range, 2.2-15.6 minutes) and slope (flow reduction rate; range, 4.4 -45.8% minute(-1)) of the decrease correlated significantly (P = 0.0001 and 0.0002, r2= 0.79 and 0.67, respectively) with treatment durability. A positive, significant (P = 0.016, r2= 0.50) association between interval time and time-to-400 mm3 was also detected in animals with depressed pre-PDT blood flow due to hydralazine administration. At 3 hours after PDT, rBF and SO2 were predictive (P < or = 0.015) of treatment durability. CONCLUSION: These data suggest a role for DCS in real-time monitoring of PDT vascular response as an indicator of treatment efficacy.

Prostaglandin E and prostacyclin receptor expression in tumor and host tissues from MCG 101-bearing mice: a model with prostanoid-related cachexia.

Preclinical and clinical studies in our laboratory have suggested that prostaglandin (PG) E2 is involved in anorexia and cachexia development, although the role of COX pathways on the pathogenesis of cancer cachexia remains to be clarified. Expressions of PGE (EP1, EP2, EP3alpha,beta,gamma and EP4) and PGI (IP) receptors in the central nervous system (brain cortex, hypothalamus and brain stem), in peripheral (liver, white adipose tissue and skeletal muscle) and tumor tissue from MCG-101-bearing mice with and without indomethacin treatment were investigated by RT-PCR and immunohistochemistry. Expression of EP1 in the liver and EP4 receptor in white adipose tissue were upregulated and responded to indomethacin treatment, while downregulated expression of EP3 in skeletal muscle from tumor-bearing mice was unresponsive to indomethacin treatment despite improved carcass weight. Expression of EP and IP receptors in brain and tumor tissue from tumor-bearing mice were neither related nor responsive to systemic PGE2 levels including increased IL-1beta, IL-6 and TNF-alpha host activities. The expression IP receptor in CNS, peripheral tissue and tumor tissue was unchanged by cachexia development. Our results suggest that transcription of EP receptors in liver, fat and skeletal muscle tissue may be a control level for host metabolic alterations during tumor progression, while overall EP and IP receptor expression in CNS did not indicate an important control level for appetite regulation in MCG 101-bearing mice despite prostanoid related anorexia.

Interleukin-15 is able to suppress the increased DNA fragmentation associated with muscle wasting in tumour-bearing rats.

Administration of interleukin-15 (IL-15) to rats bearing the Yoshida AH-130 ascites hepatoma (a tumour that induces an important cachectic response) resulted in a significant reduction of muscle wasting, both measured as muscle weight and as protein content of different types of skeletal muscle. In addition, the administration of the cytokine completely reversed the increased DNA fragmentation observed in skeletal muscle of tumour-bearing animals. Concerning the mechanism(s) involved in the anti-apoptotic effects of IL-15 on skeletal muscle, the administration of the cytokine resulted in a considerable decrease in both R1 (43%) and R2 (64%) TNF-alpha receptors (TNFRs), and therefore it may be suggested that IL-15 decreases apoptosis by affecting TNF-alpha signalling. Formation of NO could be the signalling event associated with the activation of apoptosis in muscle of tumour-bearing rats; indeed, administration of IL-15 decreased the inducible nitric oxide synthase protein levels by 73%, suggesting that NO formation and muscle apoptosis during tumour growth are related. In conclusion, IL-15 seems to be able to reduce/suppress protein loss and apoptosis related to muscle wasting during cancer cachexia in experimental animals.

Refractory hypothalamic alpha-mSH satiety and AGRP feeding systems in rats bearing MCA sarcomas.

In pre-anorectic tumor-bearing (TB: methylcholanthrene-induced sarcoma) rats, injection of alpha-melanocyte stimulating hormone (alpha-MSH) into the perifornical hypothalamus (PFH) had no significant effect on food intake at a dose (5 microg) that reduced feeding in non-TB control rats. Following the development of anorexia, injection of alpha-MSH MC3/MC4 receptor antagonists, SHU9119 (1 microg) or 4 microg agouti-related protein (AGRP), stimulated feeding in non-TB rats, while having no significant effect in TB rats. Concentrations of alpha-MSH were not altered significantly in ventromedial, dorsomedial or lateral hypothalamic areas of TB rats, and proopiomelanocortin (POMC) messenger RNA was not changed in TB rats in these hypothalamic areas. Determination of cytokines by ELISA in non-operated TB and non-TB rats revealed elevated IL-2 in plasma and hypothalamus as well as increased TNF-alpha in the hypothalamus of anorectic TB rats. IL-1B was not detectable in plasma and was not altered significantly in hypothalamus of TB rats. These results suggest that the POMC alpha-MSH satiety system is refractory in TB rats, even prior to the onset of anorexia. This change in MC3/MC4 receptor response does not appear to be secondary to alterations of endogenous alpha-MSH in TB rats. Cytokine involvement in the altered response to MC3/MC4 receptor stimulation and blockade is a possibility, since TNF-alpha and IL-2 were increased in hypothalamus of anorectic TB rats. Therefore, these results suggest major alterations in POMC neuropeptide systems in TB rats as anorexia progresses. Although these changes do not appear to have occurred due to grossly-altered concentrations of alpha-MSH, elevated cytokine activity in the hypothalamus may be an important factor. Due to the complex multi-factorial nature of feeding control, additional factors are likely to be involved in cancer anorexia.

Efficacy of systemic morphine suggests a fundamental difference in the mechanisms that generate bone cancer vs inflammatory pain.

Pain is the cancer related event that is most disruptive to the cancer patient's quality of life. Although bone cancer pain is one of the most severe and common of the chronic pains that accompany breast, prostate and lung cancers, relatively little is known about the mechanisms that generate and maintain this pain. Recently, we developed a mouse model of bone cancer pain and 16 days following tumor implantation into the intramedullary space of the femur, significant bone destruction and bone cancer pain-related behaviors were observed. A critical question is how closely this model mirrors human bone cancer pain. In the present study we show that, as in humans, pain-related behaviors are diminished by systemic morphine administration in a dose dependent fashion that is naloxone-reversible. Humans suffering from bone cancer pain generally require significantly higher doses of morphine as compared to individuals with inflammatory pain and in the mouse model, the doses of morphine required to block bone cancer pain-related behaviors were ten times that required to block peak inflammatory pain behaviors of comparable magnitude induced by hindpaw injection of complete Freund's adjuvant (CFA) (1-3mg/kg). As these animals were treated acutely, there was not time for morphine tolerance to develop and the rightward shift in analgesic efficacy observed in bone cancer pain vs. inflammatory pain suggests a fundamental difference in the underlying mechanisms that generate bone cancer vs. inflammatory pain. These results indicate that this model may be useful in defining drug therapies that are targeted for complex bone cancer pain syndromes.

Ibandronate treatment decreases the effects of tumor-associated lesions on bone density and strength in the rat.

Bisphosphonate treatment is beneficial against symptoms of metastatic bone disease, although less is known about the effect of preventative treatment schedules. We investigated the effect of various treatment regimens of the bisphosphonate, ibandronate (IB), on the preservation of bone quality in a rat model of tumor-induced osteolysis. Osteolytic Walker 256 (W256) carcinosarcoma cells were implanted into the left femur of female Sprague-Dawley rats, resulting in a 10% reduction in bone mineral density (BMD), a 16% reduction in bone density (BD), and a 26% reduction in failure load compared with the right femur 28 days after implantation. IB was administered subcutaneously in five different treatment schedules: (1) IB PRE-POST received IB for 26 days, prior to implantation of W256 cells in the medullary canal of the femur, and for 28 additional days after surgery; (2) IB PRE-POST SHAM received the same IB administration, but with a sham operation; (3) IB PRE received IB injections before W256 cell insertion only; (4) IB PRE-0 received IB injections for 26 days and was then killed to serve as a time zero control; and (5) IB POST received sham injection with saline before W256 cell insertion, and then received IB injections for 28 days until killing. Controls (TUMOR ONLY) received sham injections with saline prior to W256 cell insertion, and then for 28 additional days until killing. We used dual-energy X-ray absorptiometry (DXA) to measure distal femur BMD and bone mineral content (BMC), peripheral quantitative computed tomography (pQCT) to measure distal femur BD, and torsion testing to obtain torsional failure load. Combined preventative and interventional IB treatment best preserved bone mass and strength, although all treatment schedules resulted in significant improvement compared with untreated controls (TUMOR ONLY). The possibility of reducing or even preventing skeletal morbidity in cancer patients with a high risk of developing metastatic spreading to bone is exciting, and warrants further exploration.

VMN/LHA functional inhibition in tumor-bearing rats suggests hypothalamic involvement in cancer anorexia.

Food intake is mainly controlled in the hypothalamus via a series of functionally related nuclei, including the ventromedial nucleus of hypothalamus (VMN) and the lateral hypothalamic area (LHA). Since food intake is the product of meal number and meal size, we investigated the role of the VMN and LHA in influencing these feeding indices and in mediating cancer anorexia in tumor-bearing (TB) rats, via temporarily inhibiting VMN or LHA. Adult male Fischer-344 rats (n = 23) inoculated with 106 MCA sarcoma cells were studied. When anorexia developed, rats were randomly assigned to stereotaxically located bilateral intra-VMN or intra-LHA microinjections of the neuronal blocker colchicine (CX; n = 6 each group) or saline (n = 6 and n = 5, respectively). Non TB rats (NTB; n = 7) served as controls. Food intake and feeding indices were recorded by a computerized device. At onset of anorexia, a reduction of meal number occurred, leading to reduced food intake. After inhibition of VMN activity by CX, meal number significantly increased, so that food intake increased and almost normalized. In contrast, intra-LHA microinjection of either CX or saline resulted in reduction of meal size, leading to reduced food intake and death. Findings suggest that VMN and LHA influence meal number and meal size, respectively. Since cancer anorexia mainly results from an initial reduction of meal number and the inhibition of VMN led to an increase in meal number, the early effect of tumor growth on VMN activity may be an early step leading to reduced food intake.

Effect of hydralazine on interstitial fluid pressure in experimental tumours and in normal tissue.

Interstitial fluid pressure (IFP) has been recognised as the most important obstacle in macromolecular drug delivery to solid tumours. The aim of our study was to measure the IFP simultaneously in tumour and in muscle or in subcutis and to determine whether injection of hydralazine reduces differentially tumour IFP with respect to IFP in surrounding and normal tissues. In addition, it was of interest whether the decrease in IFP due to hydralazine depends on tumour volume and/or on initial IFP. Measurements of IFP were performed by means of the wick-in-needle technique and they were obtained on tumours of different size. In both tumour models, hydralazine significantly reduced the pretreatment IFP level. On average IFP decreased by 31% and 14% from the initial value in SAF and LPB tumours, respectively. On the contrary, hydralazine did not decrease IFP in normal tissue. Injection of NaCl solution instead of hydralazine had no effect on IFP either in tumours or in subcutis/muscle. The results of our study on the effect of hydralazine on IFP in SAF and LPB tumour model are in accordance to previously reported studies. The initial IFP in tumour is positively-correlated with the tumour size, while the decrease in the tumour IFP is independent of the initial IFP value. In addition, the decrease in tumour IFP is not correlated to tumour volume.

Clustered cancer cells show a distinct adhesion behavior from single cell form under physiological shear conditions.

It remains a question whether hematogeneous metastasis arises from a single cancer cell attached to the local endothelium or from a cluster of cancer cells trapped in the vascular bed in the target organ. Adhesive interaction of the single cell form and the clustered form of cancer cells was examined under flow conditions, using two subclones of mouse colon adenocarcinoma Colon 26. A subclone NL17, but not NL14, formed many clusters composed of tumor cells and platelets just after the addition of platelet rich plasma (PRP). Under the shear of 1.0 dyn/cm3, the clustered form of NL17 tethered on laminin or mouse endothelial cell line in hepatic sinusoids (HSE) more frequently than the single cell form of NL17 and NL14. However, all of the clusters showed only transient attachment and never underwent stable arrest on coated laminin, while the single cell form of NL14 and NL17 underwent immediate arrest under shear conditions. On HSE stimulated with TNF-alpha, a small number of NL17 clusters made stable adhesion, although all the clusters detached if the shear stress was increased above 4.0 dyn/cm2. In contrast, the single form of arrested NL17 as well as NL14 remained adherent even at shear of 8.0 dyn/cm2. Compared with single cell, binding of cancer cell clusters to laminin and HSE showed lower resistance to shear stress, although they had adhesive interactions more frequently in flow condition. Since NL17 cells form significantly more metastases by intravenous injection in vivo, our data suggest that "stable adhesion" observed in our flow assay system is not always a prerequisite for clustered cancer cells to develop into metastatic lesions.

Mouse SaI sarcoma tumor model.

SaI is an A/J-derived (H-2K(k)D(d)) transplantable mouse fibrosarcoma that produces a solid tumor when inoculated subcutaneously, intradermally, or intramuscularly, and an ascites tumor when inoculated intraperitoneally into syngeneic mice. This unit describes the establishment of primary solid SaI/N tumor, the establishment of SaI ascites tumor, and the removal for sampling of in vivo-passaged SaI ascites tumor cells in mice.

Absence of functional lymphatics within a murine sarcoma: a molecular and functional evaluation.

Despite a clinically recognized association between the lymphatics and metastasis, the biology of tumor-lymphatic interaction is not clearly understood. We report here that functional lymphatic capillaries are absent from the interior of a solid tumor, despite the presence within the tumor of the lymphangiogenic molecule vascular endothelial growth factor (VEGF)-C and endothelial cells bearing its receptor, VEGF receptor 3. Functional lymphatics, enlarged and VEGF receptor 3 positive, were detected in some tumors only at the tumor periphery (within 100 microm of the interface with normal tissue). We conclude that although lymphangiogenic factors are present, formation of functional lymphatic vessels is prevented, possibly due to collapse by the solid stress exerted by growing cancer cells.