Anti- Versus Pro-Inflammatory Metabololipidome Upon Cupping Treatment.

BACKGROUND/AIMS: This study aimed to explore the metabololipidome in mice upon cupping treatment. METHODS: A nude mouse model mimicking the cupping treatment in humans was established by administrating four cupping sets on the back skin for 15 minutes. UPLC-MS/ MS was performed to determine the PUFA metabolome in mice skin and blood before and after cupping treatment. The significantly changed lipids were administered in macrophages to assess the production of pro-inflammatory cytokines IL-6 and TNF-α by ELISA. RESULTS: The anti-inflammatory lipids, e.g. PGE1, 5,6-EET, 14,15-EET, 10S,17S-DiHDoHE, 17R-RvD1, RvD5 and 14S-HDoHE were significantly increased while pro-inflammatory lipids, e.g. 12-HETE and TXB2 were deceased in the skin or plasma post cupping treatment. Cupping treatment reversed the LPS-stimulated IL-6 and TNF-α expression in mouse peritoneal exudates. Moreover, 5,6-EET, PGE1 decreased the level of TNF-α, while 5,6-EET, 5,6-DHET downregulated IL-6 production in macrophages. Importantly, 14,15-EET and 14S-HDoHE inhibited both IL-6 and TNF-α induced by lipopolysaccharide (LPS). 17-RvD1, RvD5 and PGE1 significantly reduced the LPS-initiated TNF-α, while TXB2 and 12-HETE further upregulated the LPS-enhanced IL-6 and TNF-α expression in macrophages. CONCLUSION: Our results reveal the identities of anti-inflammatory versus pro-inflammatory metabolipidome and suggest the potential therapeutic mechanism of cupping treatment.

Analysis, physiological and clinical significance of 12-HETE: a neglected platelet-derived 12-lipoxygenase product.

While the importance of cyclooxygenase (COX) in platelet function has been amply elucidated, the identification of the role of 12-lipoxygenase (12-LOX) and of its stable metabolite, 12-hydroxyeicosatretraenoic acid (12-HETE), has not been clarified as yet. Many studies have analysed the implications of 12-LOX products in different pathological disorders but the information obtained from these works is controversial. Several analytical methods have been developed over the years to simultaneously detect eicosanoids, and specifically 12-HETE, in different biological matrices, essentially enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), high performance liquid chromatography (HPLC) and mass spectrometry coupled with both gas and liquid chromatography methods (GC- and LC-MS). This review is aimed at summarizing the up to now known physiological and clinical features of 12-HETE together with the analytical methods used for its determination, focusing on the critical issues regarding its measurement.

Fatty acids induce leukotriene C4 synthesis in macrophages in a fatty acid binding protein-dependent manner.

Obesity results in increased macrophage recruitment to adipose tissue that promotes a chronic low-grade inflammatory state linked to increased fatty acid efflux from adipocytes. Activated macrophages produce a variety of pro-inflammatory lipids such as leukotriene C4 (LTC4) and 5-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE) suggesting the hypothesis that fatty acids may stimulate eicosanoid synthesis. To assess if eicosanoid production increases with obesity, adipose tissue of leptin deficient ob/ob mice was analyzed. In ob/ob mice, LTC4 and 12-HETE levels increased in the visceral (but not subcutaneous) adipose depot while the 5-HETE levels decreased and 15-HETE abundance was unchanged. Since macrophages produce the majority of inflammatory molecules in adipose tissue, treatment of RAW264.7 or primary peritoneal macrophages with free fatty acids led to increased secretion of LTC4 and 5-HETE, but not 12- or 15-HETE. Fatty acid binding proteins (FABPs) facilitate the intracellular trafficking of fatty acids and other hydrophobic ligands and in vitro stabilize the LTC4 precursor leukotriene A4 (LTA4) from non-enzymatic hydrolysis. Consistent with a role for FABPs in LTC4 synthesis, treatment of macrophages with HTS01037, a specific FABP inhibitor, resulted in a marked decrease in both basal and fatty acid-stimulated LTC4 secretion but no change in 5-HETE production or 5-lipoxygenase expression. These results indicate that the products of adipocyte lipolysis may stimulate the 5-lipoxygenase pathway leading to FABP-dependent production of LTC4 and contribute to the insulin resistant state.

12-hydroxy-eicosatetraenoic acid (12-HETE): a biomarker of Churg-Strauss syndrome.

BACKGROUND: Churg-Strauss syndrome (CSS) shares similarities with asthma and hypereosinophilic syndrome (HES). Eicosanoids--important inflammatory and signaling molecules--are present in exhaled breath condensate (EBC) and broncho-alveolar lavage fluid (BALF). OBJECTIVES: To assess eicosanoid profile both in EBC and BALF of CSS subjects searching for a pattern characteristic of this syndrome. METHODS: EBCs from 23 CSS patients, 30 asthmatics, 12 HES patients and 54 healthy controls (HC) were assessed quantitatively for 19 eicosanoids by a high-performance liquid chromatography - tandem mass spectrometry (HPLC-MS/MS). In addition, in 21 of 23 CSS subjects and in nine asthmatics, eicosanoids were determined in BALF. RESULTS: EBC from CSS patients showed markedly elevated levels of 12-HETE as compared with other studied groups. BALF was characterized by a significant elevation of 12-HETE and its metabolite 12-tetranor HETE in CSS as compared with asthma. Clinical activity of CSS correlated with 12-HETE and its metabolites levels in BALF, but not in EBC. CONCLUSION AND CLINICAL RELEVANCE: CSS is clearly distinguished from bronchial asthma, and HES by a marked increase in 12-HETE concentration in both EBC and BALF. This points to a possible new pathogenic mechanism in CSS and may help in future in establishing the diagnosis of CSS.

Effect of fish oil on levels of R- and S-enantiomers of 5-, 12-, and 15-hydroxyeicosatetraenoic acids in mouse colonic mucosa.

The balance of putative pro- and antiinflammatory lipoxygenase (LOX)-derived S-hydroxyeicosatetraenoic acids (S-HETEs) in colon mucosa is a potential target for modulating colon cancer risk and progression. The biological effects of S-HETEs and R-hydroxyeicosatetraenoic acids (produced by distinct pathways) may differ, but levels of these compounds in the colon are unknown. The objective of this study was to develop chiral methods to characterize hydroxyeicosatetraenoic (HETE) enantiomers in colonic mucosa and evaluate the effects of fish oil on HETE formation. C57BL/6 mice (COX-1 null, COX-2 null, wild-type) were fed a diet supplemented with either olive oil or menhaden oil for 11 wk, and R-/S-HETEs in colonic mucosa were quantified by chiral LC-MS/MS. The R-enantiomer comprised 60-72% of 5-HETE, 18-58% of 15-HETE, and 1-16% of 12-HETE in colonic mucosa, suggesting that non-LOX sources contribute to HETE profiles. Fish oil reduced levels of both R- and S-HETEs, and increased the preponderance of the R-enantiomers (particularly 12- and 15-HETEs). There was apparent shunting of arachidonic acid to 12-/15-LOX in the COX-1 null animals. This is the first report of the enantiomeric composition of HETEs in the colon in vivo and shows large effects of fish oil in the normal colon.

Mass spectrometry-based quantitative analysis and biomarker discovery.

Mass spectrometry-based quantitative analysis and biomarker discovery using metabolomics approach represent one of the major platforms in clinical fields including for the prognosis or diagnosis, assessment of severity and response to therapy in a number of clinical disease states as well as therapeutic drug monitoring (TDM). This review first summarizes our mass spectrometry-based research strategy and some results on relationship between cysteinyl leukotriene (cysLT), thromboxane (TX), 12-hydroxyeicosatetraenoic acid (12-HETE) and other metabolites of arachidonic acid and diseases such as atopic dermatitis, rheumatoid arthritis and diabetes mellitus. For the purpose of evaluating the role of these metabolites of arachidonic acid in disease status, we have developed sensitive determination methods with simple solid-phase extraction and applied in clinical settings. In addition to these endogenous compounds, using mass spectrometry, we have developed actually applicable quantitative methods for TDM. Representative example was a method of TDM for sirolimus, one of the immunosuppressant agents for a recipient of organ transplant, which requires rigorous monitoring of blood level. As we recognized great potential in mass spectrometry during these researches, we have become interested in metabolomics as the non-targeted analysis of metabolites. Now, established strategy for the metabolomics investigation applies to samples from cells, animals and humans to separate groups based on altered patterns of metabolites in biological fluids and to identify metabolites as potential biomarkers discriminating groups. We would be honored if our research using mass spectrometry would contribute to provide useful information in the field of medical pharmacy.

Simultaneous quantification of arachidonic acid metabolites in cultured tumor cells using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry.

A validated method is described for the simultaneous analysis of PGE2, 11-, 12-, and 5-HETEs from cultured cells using HPLC negative electrospray ionization tandem mass spectrometry (LC/MS/MS). This method permits quantification of selected individual arachidonic acid metabolites from cell extracts without derivatization, multiple purification steps, or lengthy separation times required by traditional GC-MS- or HPLC-UV -based methods. Accuracy assessments of values calculated using this method showed deviations from nominal values were < or =15%. An average relative deviation of 7% of mean calculated values was observed for values taken on separate days. The lower limit of detection for all metabolites was 1.3 pg. The method was used to quantify arachidonic acid metabolites present in various cancer cell lines after incubation with arachidonic acid and the selective cyclooxygenase-2 inhibitor celecoxib. Results showed that the presence of celecoxib in lung cancer A549 cells reduced production of both PGE2 and 11-HETE in a concentration-dependent manner.

Ectopic alphaIIbbeta3 integrin signaling involves 12-lipoxygenase- and PKC-mediated serine phosphorylation events in melanoma cells.

Megakaryocytic genes such as alphaIIbbeta3 can be expressed by malignant cells as part of the disturbances in their gene regulation. However, the function of the gene product greatly depends on the interaction of the ectopic protein with the new environment. The outside-in signaling of the ectopically expressed alphaIIbbeta3 integrin was studied in B16a murine melanoma cells using a monoclonal antibody, specifically directed to the activated conformation of alphaIIbbeta3, PAC-1 and the physiological ligand, fibrinogen. Ligation of alphaIIbbeta3 induced down-regulation of FAK but serine phosphorylation of three protein bands, 20/21, 85 and 140 kDa within 1-15 min. Flow cytometry indicated that the ligation of the receptor in B16a cells induces approximately 50% increase in phosphoserine positive cells within 5-15 min. 12-lipoxygenase is placed downstream in the signaling pathway, since ligation of alphaIIbbeta3 induces 12-HETE production within 5 min and pretreatment of tumor cells with select lipoxygenase inhibitior, Baicalein, prevents the increase in serine phosphorylation. Confocal microscopy of adherent tumor cells demonstrated rearrangement of actin filaments upon alphaIIbbeta3 ligation paralleled by downregulation of p125FAK and phoshotyrosine+ adhesion plaques and translocation of PKCalpha to stress fibers and cortical actin. PKC appears to be the major effector serine kinase of the alphaIIbbeta3-coupled signaling pathway, since pretreatment of tumor cells with a select PKC inhibitor, Calphostin C, prevents the ligation-induced serine phosphorylation. Previous studies have indicated a role for the 12-lipoxygenase-PKC signaling pathway in platelet aggregation as well as tumor invasion, therefore the involvement of this cascade in the signaling of the ectopic alphaIIbbeta3 integrin may partially explain its role in tumor progression.

Polyphenols of cocoa: inhibition of mammalian 15-lipoxygenase.

Some cocoas and chocolates are rich in (-)-epicatechin and its related oligomers, the procyanidins. Fractions of these compounds, isolated from the seeds of Theobroma cacao, caused dose-dependent inhibition of isolated rabbit 15-lipoxygenase-1 with the larger oligomers being more active; the decamer fraction revealed an IC50 of 0.8 microM. Among the monomeric flavanols, epigallocatechin gallate (IC50 = 4 microM) and epicatechin gallate (5 microM) were more potent than (-)-epicatechin (IC50 = 60 microM). (-)-Epicatechin and procyanidin nonamer also inhibited the formation of 15-hydroxy-eicosatetraenoic acid from arachidonic acid in rabbit smooth muscle cells transfected with human 15-lipoxygenase-1. In contrast, inhibition of the lipoxygenase pathway in J774A.1 cells transfected with porcine leukocyte-type 12-lipoxygenase (another representative of the 12/15-lipoxygenase family) was only observed upon sonication of the cells, suggesting a membrane barrier for flavanols in these cells. Moreover, epicatechin (IC50 approx. 15 microM) and the procyanidin decamer inhibited recombinant human platelet 12-lipoxygenase. These observations suggest general lipoxygenase-inhibitory potency of flavanols and procyanidins that may contribute to their putative beneficial effects on the cardiovascular system in man. Thus, they may provide a plausible explanation for recent literature reports indicating that procyanidins decrease the leukotriene/prostacyclin ratio in humans and human aortic endothelial cells.

Promutagenic etheno-DNA adducts in multistage mouse skin carcinogenesis: correlation with lipoxygenase-catalyzed arachidonic acid metabolism.

Formation of the lipoxygenase-catalyzed metabolites of arachidonic acid, 8-hydroxyeicosatetraenoic acid (8-HETE) and 12-hydroxyeicosatetraenoic acid (12-HETE), and of the exocyclic DNA adducts 1,N(6)-ethenodeoxyadenosine (epsilondA) and 3, N(4)-ethenodeoxycytidine (epsilondC) was investigated in NMRI mouse skin carcinogenesis induced by 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). In reversible papillomas obtained after 20 weeks of TPA treatment, 15- and 68-fold higher contents of 8-HETE and 12-HETE, respectively, were observed, which were paralleled by 12- and 9-fold increased amounts of epsilondA and epsilondC, respectively. When compared to the level in vehicle-treated control skin, these elevations were statistically significant. In irreversible papillomas harvested 20 weeks after the last TPA treatment, the levels of HETEs and etheno-DNA adducts were found to be slightly reduced, as compared to those in reversible papillomas, but were still increased over control levels in age-matched mice. Comparison of mean group values by simple regression analysis showed a close positive correlation between HETE and etheno-DNA adduct levels. Consistent with the miscoding properties of epsilondA causing mainly A --> T transversions, its increased formation in papillomas could thus contribute to this type of mutation in codon 61 of cHa-ras, shown to be a hallmark of DMBA-initiated and TPA-promoted mouse skin carcinogenesis. Although direct evidence that etheno adducts are derived from lipoxygenase-catalyzed metabolites of arachidonic acid is missing, our results implicate DNA damage by oxidative stress and lipid peroxidation as a cause of genetic instability observed at late stages of tumor promotion in mouse skin carcinogenesis.

Quantitation of arachidonic acid metabolites in small tissue biopsies by reversed-phase high-performance liquid chromatography.

Arachidonic acid metabolites exert a variety of distinct biological effects on the initiation and resolution of inflammatory diseases and their measurements in tissue can be critical to evaluate their regulatory function during the course of inflammation and to supplement in vitro experiments. The aim of this study was the detection and quantitative analysis of four arachidonic acid metabolites in small-sized biopsies of human periodontal tissues. The biopsies were homogenized and injected directly into a single analytical column of a RP-HPLC system. Detection was performed by a photodiode array detector. Calibration was established by dilutions of authentic standards of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), and 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). A total of 38 specimens weighing between 19 and 191 mg (wet tissue) were analyzed (mean = 59.9 +/- 30.2 mg). The detection limits were 1 pg for LTB4 and 12-HETE, 0.5 pg for 15-HETE, and 10 ng for PGE2. The concentrations of PGE2 and LTB4 were significantly higher in inflamed than in healthy periodontal tissues (P = 0.0079; P = 0. 0114). 12-HETE was detected in one biopsy (30 pg/g); 15-HETE was not detected. This method of homogenization, extraction, and analysis of arachidonic acid metabolites by RP-HPLC appears to be well suited for studies of human oral biopsies. Only small tissue samples and minimal laboratory equipment were required for a sensitive analysis.

The 650-kDa 12(S)-hydroxyeicosatetraenoic acid binding complex: occurrence in human platelets, identification of hsp90 as a constituent, and binding properties of its 50-kDa subunit.

A cytosolic 650-kDa complex which binds 12(S)-hydroxy-5,8,10, 14-eicosatetraenoic acid (12(S)-HETE) with high affinity and specificity has been found in various cell lines but not until now in platelet cytosol. After incubation of human platelets with 12(S)-[3H]HETE, a labeled cytosolic 650-kDa complex was isolated. As previously shown for the binding complex in Lewis lung carcinoma (LLC) cells, ATP treatment transformed the platelet complex into a 50-kDa ligand-binding subunit. These results are of interest for two reasons: (a) 12(S)-HETE is a major arachidonic acid metabolite in platelets, and (b) platelets contain large amounts of the cell adhesion molecule GpIIb/IIIa, the activation of which is regulated by 12(S)-HETE. Hsp90 was found to be a component of the 12(S)-HETE binding complex in Lewis lung carcinoma cells, and the 50-kDa ligand-binding subunit itself bound 12(S)-HETE with high affinity. Competition experiments showed that 12(R)-HETE, 15-deoxy-Delta12, 14-prostaglandin J2, and 5(S)-HETE had lower affinity for the 50-kDa subunit than 12(S)-HETE. The 12(S)-HETE binding protein appears to be distinct from known members of the steroid hormone receptor superfamily of nuclear receptors.

Antiplatelet effects of conjugated linoleic acid isomers.

Conjugated diene isomers of linoleic acid (CLA) are normal constituents of certain foods and exhibit anticarcinogenic and antiatherogenic properties. In the present study, the effects of several CLA isomers on human platelet aggregation and arachidonic acid metabolism were examined. It was found that 9c,11t-CLA, 10t, 12c-CLA and 13-hydroxy-9c,11t-octadecadienoic acid (13-HODE) inhibited arachidonic acid- and collagen-induced platelet aggregation with I50s in the 5-7 microM range. The nonconjugated 9c, 12c-LA was about 300% and 50%, respectively, less potent an inhibitor with these aggregating agents. Using either thrombin or the calcium ionophore A23187 as aggregating agents, a CLA isomer mix was also found to be more inhibitory than 9c,12c-LA. The 9c,11t- and 10t,12c-CLA isomers as well as the CLA isomer mix inhibited formation of the proaggregatory cyclooxygenase-catalyzed product TXA2, as measured by decreased production of its inactive metabolite [14C]TXB2 from exogenously added [14C]arachidonic acid (I50s=9-16 microM). None of the CLA isomers tested inhibited production of the platelet lipoxygenase metabolite [14C]12-HETE. The additional presence of a hydroxyl group gave opposite results: 13-HODE (I50=3 microM) was about 4-fold more potent a cyclooxygenase inhibitor than the 9c,11t-CLA isomer but 9-HODE was 2- to 3-fold less effective an inhibitor (I50=34 microM) of [14C]TXB2 formation than the corresponding 10t,12c-CLA. In both the aggregation and arachidonic acid metabolism experiments, the inhibitory effects of CLA on platelets were reversible and dependent on the time of addition of either the aggregating agent or the [14C]arachidonic acid substrate. These studies suggest that CLA isomers may also possess antithrombotic properties.

LTB4 as marker of 5-LO inhibitory activity of two new N-omega-ethoxycarbonyl-4-quinolones.

The supposed 5-LO inhibitory activity of two N-omega-ethoxycarbonyl-4-quinolones was tested determining leukotriene B4 (LTB4) in RBL-1 cell cultures, pretreated with the two compounds of interest. LTB4, obtained by solid-phase extraction (SPE) from cell cultures supernatants, was determined by micellar electrokinetic chromatography (MEKC). The analysis was performed using an uncoated capillary, filled with borate buffer at pH 8.3, containing 12.5 mM SDS as micelles generator. Therefore, following the decreasing of LTB4 it was possible to verify the 5-LO inhibitory activity of two quinolone derivatives. To asses the suitability of the use of LTB4 as marker of the activity of the new compounds, the analysis was repeated using quercetin, a well known 5-LO inhibitor.

Development of smokeless tobacco-induced oral mucosal lesions.

This study examined clinical and inflammatory mediator parameters during the development of snuff-induced mucosal lesions. Nineteen smokeless tobacco (ST) users placed moist snuff at designated new placement sites over either a 2- or 7-day period. By day 2, the predominant clinical alteration was an erythematous reaction, and one-third of the subjects demonstrated white striations in combination with erythema or ulceration. By 7 days, 56% of the subjects displayed white striated lesions. Tissue concentrations (pg/mg) of IL-1beta were 4.6+/-1.6 at new placement sites as compared with 0.7+/-0.4 at control sites (P<0.05). PGE2 and IL-1alpha concentrations also were significantly higher (P<0.05) at new placement sites as compared with control sites (9.4+/-2.2 vs 4.1+/-0.7 and 6.2+/-1.3 vs 3.2+/-0.7, respectively). In view of the recognized role of PGE2 and IL-1 in immune and inflammatory functions, these mediators may play a role in the pathogenesis of clinical alterations at ST placement sites.

Aspirin enhances platelet-derived growth factor-induced vascular smooth muscle cell proliferation.

PURPOSE: Aspirin is frequently used after vascular reconstruction to pharmacologically prevent graft occlusion and to suppress the development of myointimal hyperplasia in vascular surgery, but its efficacy is controversial. The purpose of this study was to examine the direct effects of aspirin on platelet-derived growth factor (PDGF)-induced vascular smooth muscle cell (SMC) proliferation. METHODS: Human aortic SMCs were grown to confluence in 96 well plates. 3 x 10(-5) mol/L aspirin was added 24 hours previously and PDGF 10 ng/ml at the beginning of each experiment. Cell proliferation at 48 hours was determined using tritiated thymidine uptake. Supernatant 12-L-hydroxy 5,8,10,14-eicosatetraenoic acid (12-HETE) and prostaglandin E2 (PGE2) were measured by competitive enzyme immunoassay. RESULTS: Aspirin did not change vascular SMC proliferation rates relative to controls (4665 +/- 181 counts per minute [CPM] vs 4749 +/- 155 CPM). However, aspirin pretreatment of PDGF-stimulated vascular SMCs increased proliferation (9408 +/- 237 CPM vs 7283 +/- 283 CPM; p < 0.001). 5,8,10,14-eicosatriynoic acid, a 12-lipoxygenase inhibitor, decreased basal (2037 +/- 181 CPM vs 2306 +/- 158 CPM; p < 0.05) and PDGF-stimulated vascular SMC proliferation (4909 +/- 1089 CPM vs 4310 +/- 1022 CPM; p < 0.001). Aspirin increased supernatant 12-HETE levels and decreased PGE2 levels in both basal and PDGF-stimulated cell cultures. CONCLUSIONS: Aspirin enhances PDGF-stimulated vascular SMC proliferation. The effects of aspirin on vascular SMC proliferation may be mediated by changes in vascular SMC arachidonic acid metabolism.

Role of arachidonic acid metabolites in tumor growth inhibition by nonsteroidal antiinflammatory drugs.

PURPOSE: A murine model of squamous cell carcinoma (SCC) was used to determine the role of arachidonic acid (AA) metabolites in the growth of SCC of the head and neck. MATERIALS AND METHODS: C3H/HeJ mice bearing SCC (SCC VII) were treated with cyclooxygenase inhibitors (piroxicam and nabumetone) or a 5-lipoxygenase inhibitor (ketoconazole). Growth curves were established, and final tumor weights were measured. Following sacrifice, tumor tissue homogenates were assayed for prostaglandin E2 (PGE2) and 12-hydroxyeicosatetraenoic acid (12-HETE) by enzyme-linked immunosorbent assay (ELISA), and leukotriene B4 (LTB4) by radioimmunoassay (RIA). Inflammatory cell infiltrate was assessed histologically. RESULTS: A significant inhibition of tumor growth (P = .001) and final tumor weight (P = .002) was noted in mice treated with piroxicam and nabumetone. Inhibition of tumor growth was associated with increased tumor tissue levels of PGE2 (P = .04) and lymphocytic infiltration (P = .07). Significant inhibition of tumor growth (P = .002) and final tumor weight (P = .05) was also noted in mice treated with ketoconazole. CONCLUSION: These data suggest that both cyclooxygenase and lipoxygenase metabolites of AA affect tumor growth in this model and that inhibition of tumor growth by inhibitors of AA metabolism may be caused by an enhanced inflammatory cell response at the tumor site.