Enhanced FGFR signalling predisposes pancreatic cancer to the effect of a potent FGFR inhibitor in preclinical models.

BACKGROUND: Fibroblast growth factor receptor (FGFR) signalling has been implicated in pancreas carcinogenesis. We investigated the effect of FGFR inhibition in pancreatic cancer in complementary cancer models derived from cell lines and patient-derived primary tumour explants. METHODS: The effects of FGFR signalling inhibition in pancreatic cancer were evaluated using anti-FRS2 shRNA and dovitinib. Pancreatic cancers with varying sensitivity to dovitinib were evaluated to determine potential predictive biomarkers of efficacy. Primary pancreatic explants with opposite extreme of biomarker expression were selected from 13 tumours for in vivo dovitinib treatment. RESULTS: Treatment with anti-FRS2 shRNA induced significant in vitro cell kill in pancreatic cancer cells. Dovitinib treatment achieved similar effects and was mediated by Akt/Mcl-1 signalling in sensitive cells. Dovitinib efficacy correlated with FRS2 phosphorylation status, FGFR2 mRNA level and FGFR2 IIIb expression but not phosphorylation status of VEGFR2 and PDGFRß. Using FGFR2 mRNA level, a proof-of-concept study using primary pancreatic cancer explants correctly identified the tumours' sensitivity to dovitinib. CONCLUSION: Inhibiting FGFR signalling using shRNA and dovitinib achieved significant anti-cancer cancer effects in pancreatic cancer. The effect was more pronounced in FGFR2 IIIb overexpressing pancreatic cancer that may be dependent on aberrant stimulation by stromal-derived FGF ligands.

Using extracellular biomarkers for monitoring efficacy of therapeutics in cancer patients: an update.

Rapidly detectable and easily accessible markers of tumor cell death are needed for evaluating early therapeutic efficacy for immunotherapy and chemotherapy so that patients and their physicians can decide whether to remain with a given therapeutic strategy. Currently, image-based tests such as computed tomography scans and magnetic resonance imaging are used to visualize the response of a patient's tumor, but often these evaluations are not conducted for weeks to months after treatment begins. While serum levels of secreted proteins such as carcinoembryonic antigen and prostate specific antigen are commonly monitored to gauge tumor status during therapy and between image evaluations, the levels of these proteins do not always correlate well with the actual tumor response. In laboratory studies, it has been shown that tumor cells undergoing apoptosis can release cellular components into cell culture media such as cytochrome c, nucleosomes, cleaved cytokeratin-18 and E-cadherin. Studies of patient sera have found that these and other macromolecules can be found in circulation during cancer therapy, providing a potential source of material for monitoring treatment efficacy. In the future, analysis of biofluids from severe combined immunodeficiency mice bearing patient tumor specimens treated with a targeted therapy such as Apo2L/tumor necrosis factor-related apoptosis-inducing ligand will be useful in the preclinical identification of therapy response markers. In this review, the current status of the identification of serum markers of tumor cell apoptosis is provided, as well as a discussion of critical research questions that must be addressed and the considerations necessary when identifying a marker that reflects true clinical outcome.

Engineering secretable forms of chaperones for immune modulation and vaccine development.

Heat shock proteins are present in almost all intracellular compartments and serve by folding newly synthesized proteins, disassembling unstable proteins, and assisting in the transportation of proteins within the cell. Under certain circumstances they are also present on the cell surface, and can be shed or secreted into the extracellular environment. Although they possess many functional roles, their ability to stimulate innate and antigen-specific immunity have made them attractive candidates for vaccine development. Here, we review some of the approaches that have been used to genetically engineer molecular chaperones for their secretion from tumor cells or targeting them to the plasma membrane of such cells in order to promote anti-tumor responses. Treatment of tumor cells engineered to secrete or display chaperones may be of benefit, particularly in the area of cell-based vaccine development.

Thermal regulation of dendritic cell activation and migration from skin explants.

Dendritic cells (DCs) in the skin rapidly take up antigen and migrate out of the skin to draining lymph nodes for antigen presentation. As a result, these cells play an important role in generating specific immune responses against infectious agents that enter the skin and against antigens delivered as vaccines. Previous efforts revealed that fever-like elevations in body temperature enhance antigen-dependent immune responses initiated at the site of the skin and stimulate the migration of epidermal DCs to draining lymph nodes. Collectively, these data have led to the hypothesis that the activation of epidermal DCs is sensitive to physiological thermal stimuli. In this study, ear skin explants derived from BALB/c mice were either maintained at 37 degrees C or incubated at 40 degrees C for the first 6.5 h before being placed at 37 degrees C. This heating protocol altered the density and morphology of the epidermal DCs in a manner suggestive of an increased kinetics of activation-associated DC migration. Flow cytometric analysis of the emigrated cells also indicated that mild heating enhanced the migration kinetics of DCs and increased the DC expression of MHC class II and the activation marker CD86. Importantly, these migrated cells displayed higher stimulatory capacity in a mixed lymphocyte reaction compared to those of controls. Overall, these results suggest that mild thermal stimuli can enhance DC activation and function and that strategic applications of heat could enhance the potency of vaccines consisting of relatively weak antigens, such as cancer vaccines.

A phase I study of fever-range whole body hyperthermia (FR-WBH) in patients with advanced solid tumours: correlation with mouse models.

Various studies in animal tumour models have revealed the potential of fever-range whole body hyperthermia (FR-WBH) to be used in cancer therapy. To determine the safety of FR-WBH treatment in the clinic, patients with advanced solid tumours were heated in the outpatient setting to 39-39.5 degrees C for 3 or 6h, or 39.5-40 degrees C for 6h using the Heckel-HT 2000 apparatus. These WBH treatments were well tolerated, with no significant adverse events related to cardiac, hepatic, renal or pulmonary systems. In the majority of patients, flow cytometric analysis of peripheral blood leukocyte populations indicated that there were transient decreases in the number of circulating T lymphocytes and a concomitant decrease in the number of L-selectin positive lymphocytes in the peripheral blood. These findings closely mimic the affects seen previously in pre-clinical murine studies in which this same fever-like treatment was shown to inhibit tumour growth. These studies have established the safety of this treatment and will allow for future clinical trials where application of FR-WBH treatment can be combined with other anti-cancer therapies, including immunotherapy and chemotherapy.

Induction of stress proteins in a panel of mouse tissues by fever-range whole body hyperthermia.

When exposed to environmental stress, cell survival is supported by the upregulation of stress proteins such as heat shock proteins (HSPs) or glucose regulated proteins (GRPs), which help prevent protein denaturation. To begin to characterize the ability of a physiologically relevant heat exposure to induce stress protein expression, the cerebellum, cerebrum, colon, heart, kidney, liver, lung, lymph nodes, muscle, serum and thymus were extracted from BALB/c mice at various times after fever-range whole body hyperthermia (FR-WBH, 39.5-40 degrees C for 6 h) treatment. The expression of three stress proteins, HSP70, HSP110 and GRP170, was determined in these tissues and serum and compared to constitutive levels in control tissues and serum using Western analysis. Constitutive expression of GRP170 was not affected by FR-WBH in any tissue. In contrast, FR-WBH did enhance HSP expression: HSP70 in heart, kidney, lung, lymph nodes and thymus; and HSP110 in lung, lymph nodes and thymus. The lymphoid tissues displayed the most consistent upregulation of both HSP70 and HSP110 upon FR-WBH treatment. The apparent sensitivity of immunologically relevant tissues to FR-WBH may relate to the enhanced immune responses that are observed during febrile temperatures.

Recombinant CD40 ligand therapy has significant antitumor effects on CD40-positive ovarian tumor xenografts grown in SCID mice and demonstrates an augmented effect with cisplatin.

CD40 is a member of the tumor necrosis factor receptor family and was first identified with a monoclonal antibody raised against bladder carcinoma. Recombinant human CD40L has been shown previously to have a direct antitumor effect on an ovarian cancer cell line and ovarian carcinoma cells isolated from ascites fluid. We show here that rhuCD40L inhibits the growth of several ovarian adenocarcinomas derived from surgical specimens and grown as xenografts in severe combined immunodeficient mice. Two 14-day treatment cycles were more effective than one. This effect is apparently not mediated by natural killer cells, because blocking natural killer cell activity by antiasialo GM-1 did not diminish this effect. In addition to suppression of tumor growth, treatment with rhuCD40L resulted in an increased expression of FasL, an increase in apoptosis, and histological changes including increased fibrosis and areas of tumor destruction. Using this model, we examined the efficacy of rhuCD40L in combination with chemotherapeutic agents. The antitumor effect of rhuCD40L in combination with 4 mg/kg cisplatin (CDDP) was increased over the effect of CDDP alone. Furthermore, rhuCD40L increased the efficacy of a suboptimal dose of CDDP (2mg/kg) such that it matched that of high-dose CDDP alone. These data suggest a role for rhuCD40L therapy in combination with platinum based regimens for primary treatment of epithelial ovarian tumors.

Regulatory potential of fever-range whole body hyperthermia on Langerhans cells and lymphocytes in an antigen-dependent cellular immune response.

The febrile response is one of the most common features of infection and inflammation. However, temperature is rarely a variable in experimental immunological investigations. To determine whether the thermal microenvironment has any immunoregulatory potential in an Ag-dependent response, we applied a mild fever-range whole body hyperthermia (FR-WBH) protocol to BALB/c mice experiencing the contact hypersensitivity (CHS) reaction. We observed that the timing of this FR-WBH treatment relative to the different phases of the CHS response was crucial to the outcome. FR-WBH treatment before sensitization with a 0.5% FITC solution resulted in a depressed CHS response. This appears to be due to direct effects of FR-WBH on epidermal Langerhans cell trafficking to the draining lymph nodes. In contrast, application of FR-WBH directly after application of the elicitation dose of FITC solution resulted in an enhanced reaction. This result correlates with increased homing of lymphocytes to the site of elicitation. Overall, these data have important implications regarding the role of thermal changes experienced during infection and the clinical use of FR-WBH relative to immunotherapeutic strategies.

Fever-range hyperthermia dynamically regulates lymphocyte delivery to high endothelial venules.

Fever is associated with increased survival during acute infection, although its mechanism of action is largely unknown. This study found evidence of an unexpectedly integrated mechanism by which fever-range temperatures stimulate lymphocyte homing to secondary lymphoid tissues by increasing L-selectin and alpha4beta7 integrin-dependent adhesive interactions between circulating lymphocytes and specialized high endothelial venules (HEV). Exposure of splenic lymphocytes in vivo to fever-like whole-body hyperthermia (WBH; 39.8 +/- 0.2 degrees C for 6 hours) stimulated both L-selectin and alpha4beta7 integrin-dependent adhesion of lymphocytes to HEV under shear conditions in lymph nodes and Peyer patches. The adhesiveness of HEV ligands for L-selectin and alpha4beta7 integrin (ie, peripheral lymph node addressin and mucosal addressin cell adhesion molecule-1) also increased during WBH or febrile responses associated with lipopolysaccharide-induced or turpentine-induced inflammation. Similar increases in HEV adhesion occurred during hyperthermia treatment of lymph node and Peyer patch organ cultures in vitro, indicating that the local lymphoid tissue microenvironment is sufficient for the hyperthermia response. In contrast, WBH did not augment adhesion in squamous endothelium of nonlymphoid tissues. Analysis of homing of alpha4beta7(hi) L-selectin(lo) murine TK1 cells and L-selectin(hi) alpha4beta7 integrin-negative 300.19/L-selectin transfectant cells showed that fever-range temperatures caused a 3- to 4-fold increase in L-selectin and alpha4beta7 integrin-dependent trafficking to secondary lymphoid tissues. Thus, enhanced lymphocyte delivery to HEV by febrile temperatures through bimodal regulation of lymphocyte and endothelial adhesion provides a novel mechanism to promote immune surveillance.

HLA class I antigen cell surface expression is preserved on acute myeloid leukemia blasts at diagnosis and at relapse.

Human leukocyte antigens (HLA) class I molecules restrict the interaction between cytotoxic T cells and target cells. Abnormalities in HLA class I antigen expression and/or function may provide tumor cells with a mechanism for escaping immune surveillance and resisting T cell-based immunotherapies. The potential for applying T cell-based immunotherapy in the treatment of acute myeloid leukemia (AML) has stimulated interest in analyzing HLA class I antigen expression on leukemic blasts in this disease. Little information is available in the literature. We have analyzed HLA class I antigen expression on bone marrow samples from 25 newly diagnosed AML patients by indirect immunofluorescence staining with monoclonal antibodies. Five of these patients were also studied at relapse. Leukemic blasts were resolved from normal lymphocytes by staining with antiCD45 antibody; CD45 expression is dim on leukemia cells, but bright on lymphocytes. HLA class I antigen expression was higher on leukemic blasts than on autologous lymphocytes in all but one case. Moreover, there was no significant change in HLA class I antigen expression at relapse. These results suggest that abnormalities in HLA class I antigens are infrequent in AML and should not represent a major obstacle to the application of T cell-based immunotherapies in this disease.

Characterization of heat shock protein 110 and glucose-regulated protein 170 as cancer vaccines and the effect of fever-range hyperthermia on vaccine activity.

Several studies have confirmed that certain stress proteins can function as potent vaccines against a specific cancer when purified from the same tumor. Recent studies of two long-recognized but unstudied stress proteins, heat shock protein (hsp) 110 and glucose-regulated protein (grp) 170, have shown them to be efficient peptide chain-binding proteins. The present investigation examines the vaccine potential of hsp110 and grp170. First, it is shown that prior vaccination with hsp110 or grp170 purified from methylcholanthrene-induced fibrosarcoma caused complete regression of the tumor. In a second tumor model, hsp110 or grp170 purified from Colon 26 tumors led to a significant growth inhibition of this tumor. In addition, hsp110 or grp170 immunization significantly extended the life span of Colon 26 tumor-bearing mice when applied after tumor transplantation. A tumor-specific cytotoxic T lymphocyte response developed in the mice immunized with tumor-derived hsp110 or grp170. Furthermore, treatments of the mice with bone marrow-derived dendritic cells pulsed with these two proteins from tumor also elicited a strong antitumor response. Last, we showed that mild, fever-like hyperthermic conditions enhance the vaccine efficiency of hsp110 as well as heat shock cognate 70, but not grp170. These studies indicate that hsp110 and grp170 can be used in hsp-based cancer immunotherapy, that Ag-presenting dendritic cells can be used to mediate this therapeutic approach, and that fever-level hyperthermia can significantly enhance the vaccine efficiency of hsps.

Regulatory effects of fever-range whole-body hyperthermia on the LPS-induced acute inflammatory response.

The thermal component of fever is one of the most poorly understood aspects of inflammation. To evaluate the role of fever-range hyperthermia on acute inflammation, BALB/c and C57BL/6 mice were exposed to mild, long-duration whole-body hyperthermia (WBH), and serum concentrations of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), IL-1beta, and the acute phase proteins (APPs) alpha1-acid glycoprotein and haptoglobin were analyzed. WBH alone did not affect serum concentrations of these cytokines or APPs when compared with controls. In contrast, when WBH was applied just after intraperitoneal administration of lipopolysaccharide (LPS), serum concentrations of TNF-alpha and IL-6 were greater than or equal to threefold higher in BALB/c mice compared with LPS-treated controls. LPS-induced IL-6 levels were also enhanced in WBH-treated C57BL/6 mice. However, APP levels were prolonged only in WBH-treated BALB/c mice. It is interesting that in vitro hyperthermia treatment of LPS-stimulated peritoneal cells resulted in decreased cytokine production compared with controls. These results suggest that fever-range hyperthermia regulates acute inflammation in a mouse strain-specific manner that is more complex than that observed in vitro.

Flt-3 ligand inhibits growth of human ovarian tumors engrafted in severe combined immunodeficient mice.

OBJECTIVE: The current study evaluated the effects of Flt-3 ligand (FL) on the growth of human malignant ovarian tumors engrafted in severe combined immunodeficient (SCID) mice with particular attention directed at FL's effect on the host natural killer (NK) cell response against ovarian cancer xenografts. METHODS: Equal portions of surgical specimen-derived human ovarian carcinomas were engrafted subcutaneously (SC) into SCID mice. Mice were placed into one of two treatment groups 7 days after the day of implantation. Group 1 received placebo injections SC from Day 1 to Day 20 and group 2 received FL at 10 microg/day SC from Day 1 to Day 20. NK cell depletion was performed on three additional mice from group 2 starting on Day 0 using anti-asialo GM1. Serial tumor volumes were measured. On Day 21, mice from each group were sacrificed, and tumors and spleens were evaluated. Data analysis included chi(2) tests, Student t tests, and analyses of variance when appropriate. RESULTS: FL resulted in tumor growth delay compared with control (P = 0.036). When NK cell activity was depleted prior to FL administration, no tumor growth delay was observed. Spleens from FL-treated mice were larger (P < 0.01) with expanded white pulp compared with controls. Histologic examination of tumor sections from FL-treated mice revealed regions of solid tumor growth with glandular architecture similar to that seen in control tumors; however, there was an obvious increase in regions composed largely of dense fibrosis in the FL-treated tumors. NK cells and other infiltrating cells could be detected in clusters among tumors from mice treated with FL whereas these cells were only occasionally detected in sections of control tumors. CONCLUSION: FL treatment resulted in an antitumor response against human ovarian cancer engrafted in SCID mice and this inhibition appears to be largely host NK cell mediated. The tumor inhibition seen in this model is similar to that previously seen using syngeneic tumors grown in an immunocompetent animal model. Results from this model can potentially be extrapolated to treatment of human ovarian cancer patients.

Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors.

BACKGROUND: Inherited mutations in the BRCA1 gene may be responsible for almost half of inherited breast carcinomas. However, somatic (acquired) mutations in BRCA1 have not been reported, despite frequent loss of heterozygosity (LOH or loss of one copy of the gene) at the BRCA1 locus and loss of BRCA1 protein in tumors. To address whether BRCA1 may be inactivated by pathways other than mutations in sporadic tumors, we analyzed the role of hypermethylation of the gene's promoter region. METHODS: Methylation patterns in the BRCA1 promoter were assessed in breast cancer cell lines, xenografts, and 215 primary breast and ovarian carcinomas by methylation-specific polymerase chain reaction (PCR). BRCA1 RNA expression was determined in cell lines and seven xenografts by reverse transcription-PCR. P values are two-sided. RESULTS: The BRCA1 promoter was found to be unmethylated in all normal tissues and cancer cell lines tested. However, BRCA1 promoter hypermethylation was present in two breast cancer xenografts, both of which had loss of the BRCA1 transcript. BRCA1 promoter hypermethylation was present in 11 (13%) of 84 unselected primary breast carcinomas. BRCA1 methylation was strikingly associated with the medullary (67% methylated; P =.0002 versus ductal) and mucinous (55% methylated; P =.0033 versus ductal) subtypes, which are overrepresented in BRCA1 families. In a second series of 66 ductal breast tumors informative for LOH, nine (20%) of 45 tumors with LOH had BRCA1 hypermethylation, while one (5%) of 21 without LOH was methylated (P =.15). In ovarian neoplasms, BRCA1 methylation was found only in tumors with LOH, four (31%) of 13 versus none of 18 without LOH (P =.02). The BRCA1 promoter was unmethylated in other tumor types. CONCLUSION: Silencing of the BRCA1 gene by promoter hypermethylation occurs in primary breast and ovarian carcinomas, especially in the presence of LOH and in specific histopathologic subgroups. These findings support a role for this tumor suppressor gene in sporadic breast and ovarian tumorigenesis.

Comparison of the effects of two different whole body hyperthermia protocols on the distribution of murine leukocyte populations.

Two predominant WBH protocols presently being used in clinical trials include a low temperature, long duration (LL) WBH, where core body temperature is raised to 39.5-40 degrees C for 6h or more, and a high temperature, short duration (HS) WBH, where core body temperature is raised to 41.8 degrees C for up to 2h. Here, the effects of LL-WBH and HS-WBH on leukocyte populations in the blood, spleen, lymph node (LN) and peritoneal cavity (PerC) of Balb/c mice were compared using flow cytometry. The total numbers of peripheral blood leukocytes decreased up to 2-fold immediately after LL-WBH, reflecting a decrease of lymphocyte numbers compared to controls. In contrast, the numbers of blood leukocytes are increased 2.7-fold immediately after HS-WBH compared to controls, reflecting an increase in lymphocytes, monocytes and granulocytes. After both LL- and HS-WBH treatment, leukocyte numbers in the spleen are decreased approximately 2-fold, again reflecting decreases in lymphocyte numbers. In the PerC, total numbers of leukocytes are also significantly decreased (2-fold) during LL-WBH but not HS-WBH. Total numbers of leukocytes in the LNs were unaffected by both LL- and HS-WBH. Overall, these data reveal differential effects of the LL- and HS-WBH protocols on leukocyte populations in the blood, spleen, LN and PerC of Balb/c mice.

Regulation of immune activity by mild (fever-range) whole body hyperthermia: effects on epidermal Langerhans cells.

Inflammation of the skin and systemic fever, both of which occur with injury or infection, include a hyperthermic component that many believe constitutes a physiological stress. Such increases in local or systemic body temperature may also have a regulatory effect on immune function. Langerhans cells (LCs), the dendritic cells of the skin, continuously monitor the extracellular matrix of the skin by taking up particles and microbes that they then carry to draining lymph nodes for presentation to T lymphocytes. We hypothesize that the thermal element of inflammation and/or fever may help regulate the activation and migration of LCs out of the epidermis. To test this hypothesis, Balb/ c mice were exposed to a mild (39.8 degrees C +/- 0.2 degrees C), long-duration (6 hours) whole body hyperthermia (WBH) treatment, which mimics the thermal component of fever. The number of LCs and their morphology were analyzed at various time points up to 7 days after the initiation of WBH. The LCs of the ear epidermis were visualized using a fluorescein isothiocyanate-conjugated antibody specific for the major histocompatibility complex (MHC) class II molecule and confocal microscopy. Although MHC class II staining was diffuse on the surface of the LC body and dendritic extensions of both WBH and control samples, the WBH-treated LCs exhibited a more punctate morphology with fewer dendritic processes compared with control LCs. A significant decrease in the number of LCs was also observed 1 to 5 days after WBH treatment. Furthermore, in vitro heating of Balb/c ear skin cultures at 40 degrees C for 6 to 8 hours enhanced the numbers of viable LCs that migrated into the culture wells. These results suggest that WBH treatment stimulates epidermal LCs in the absence of foreign antigen.

Polarized expression of immunoglobulin, spectrin, and protein kinase C beta II occurs in B cells from normal BALB/c, autoimmune lpr, and anti-ssDNA transgenic, tolerant mice.

The rapid redistribution of B cell surface immunoglobulin to a cap upon cross-linking treatment is a well-described phenomenon, the physiological significance of which is unknown. We describe the observation that splenic B cells from unimmunized normal, autoimmune, and tolerant mice express naturally occurring capped immunoglobulin in the absence of exogenous stimulation. The percentage of capped B cells increases to 20% of B cells by age 16 weeks in the progressive autoimmune lpr mouse. Transgenic, tolerant mice expressing lpr-derived genes for ssDNA-binding antibody also demonstrate a large percentage (35-75%) of immunoglobulin-capped splenic B cells. In these capped B cells, protein kinase C beta II, the cytoskeletal proteins spectrin and ankyrin, and the lipophilic probe diI are enriched beneath the site of the immunoglobulin cap. These data suggest that polarization of surface receptors, signaling molecules, anionic phospholipid domains, and cytoskeletal proteins may be an important part of the B cell immune response in vivo.

Characterization of mild whole-body hyperthermia protocols using human breast, ovarian, and colon tumors grown in severe combined immunodeficient mice.

OBJECTIVE: We have shown that one treatment of fever-like whole body hyperthermia (WBH) on mice bearing human breast tumors results in a tumor growth delay. Our goal was to repeat this study in mice bearing human ovarian or colon tumors. We further evaluated this WBH protocol by performing multiple and interrupted WBH treatments. METHODS: Human tumors were grown in severe combined immunodeficient (SCID) mice. For WBH, core body temperatures were maintained at 39.8+/-0.2 degrees C for 6-8 hours. Multiple treatments were given 6-7 days apart. Interrupted WBH consisted of three 2-hour heatings, 15 minutes apart. Tumor growth time (TGT) was the number of days to grow 1.5 or 2 times in volume. RESULTS: For WBH-treated ovarian tumors, TGT was 12+/-1.2d, compared with 5.0+/-0.1d for untreated mice (P < 0.05). For colon tumors with one WBH treatment TGT was 4.4+/-1.1d. Two and three treatments had TGTs of 9+/-2.3d and 8+/-1.6d. For the untreated tumors, TGT was 2+/-0.7d (P < 0.01 for one, two, and three treatments). Histological examination indicated that one and two treatments were associated with cellular damage within the tumors. With a slower growing colon tumor, the TGT was 24+/-3.3d with three WBH treatments, compared with 14+/-1.8d for controls (P < 0.01). The TGT of breast tumors treated with interrupted WBH was not significantly different than the noninterrupted, with TGT of 7.3+/-0.8d and 6.2+/-1.0d, respectively. CONCLUSIONS: These data illustrate that WBH causes a tumor growth delay in mice bearing human ovarian and colon tumors. This response is enhanced with a second treatment of WBH. Interrupted and noninterrupted WBH give comparable anti-tumor results. We will continue to evaluate WBH in various animal models to optimize its potential for clinical administration and maximize the anti-tumor response.

Effect of fever-like whole-body hyperthermia on lymphocyte spectrin distribution, protein kinase C activity, and uropod formation.

Regional inflammation and systemic fever are hallmarks of host immune responses to pathogenic stimuli. Although the thermal element of fever is thought to enhance the activity of immune effector cells, it is unclear what the precise role of increased body temperatures is on the activation state and effector functions of lymphocytes. We report here that mild, fever-like whole body hyperthermia (WBH) treatment of mice results in a distinct increase in the numbers of tissue lymphocytes with polarized spectrin cytoskeletons and uropods, as visualized in situ. WBH also induces a coincident reorganization of protein kinase C (PKC) isozymes and increased PKC activity within T cells. These hyperthermia-induced cellular alterations are nearly identical with the previously described effects of Ag- and mitogen-induced activation on lymphocyte spectrin and PKC. Immunoprecipitation studies combined with dual staining and protein overlay assays confirmed the association of PKC beta and PKC theta with spectrin following its reorganization. The receptor for activated C kinase-1 was also found to associate with the spectrin-based cytoskeleton. Furthermore, all these molecules (spectrin, PKC beta, PKC theta, and receptor for activated C kinase-1) cotranslocate to the uropod. Enhanced intracellular spectrin phosphorylation upon WBH treatment of lymphocytes was also found and could be blocked by the PKC inhibitor bisindolylmaleimide I (GF109203X). These data suggest that the thermal element of fever, as mimicked by these studies, can modulate critical steps in the signal transduction pathways necessary for effective lymphocyte activation and function. Further work is needed to determine the cellular target(s) that transduces the signaling pathway(s) induced by hyperthermia.