Comparisons among pimonidazole binding, oxygen electrode measurements, and radiation response in C3H mouse tumors.

Pimonidazole binding was compared with oxygen electrode measurements and with measurements of the radiobiologically hypoxic fraction in C3H mammary tumors in which oxygenation was manipulated by means of subjecting tumor-bearing CDF1 mice to air breathing, carbogen breathing, oxygen breathing, hydralazine injection or tumor clamping. Hypoxia measured by pimonidazole binding could be correlated with both pO2 (r2 = 0.81) and radiobiologically hypoxic fraction (r2 = 0.85) in this system. The scope and limitation of pimonidazole as an immunohistochemical marker for tumor hypoxia is discussed.     

Effects of partial hepatectomy on the growth characteristics and hypoxic fractions of xenografted DLD-2 human colon cancers.

A recent publication by Leith et al. (Cancer Res. 51, 4111-4113, 1991) showed that administration of epidermal growth factor (EGF) (0.25 mg/kg; q.i.d. x 7) to mice bearing xenografted A431 human epidermoid cancers produced increased tumor growth and a reduction of the percentage of hypoxic cells within neoplasms. In contrast, sialoadenectomy, which removes the majority of circulating EGF in the mouse, resulted in slower tumor growth and increased hypoxic percentages. To investigate such homeostatic mechanisms further, we investigated the growth characteristics and hypoxic fractions of xenografted DLD-2 human colon tumors when tumor-bearing mice received partial hepatectomy (about 65%), unilateral nephrectomy, or nonspecific surgical trauma (laparotomy). Surgical procedures were performed when tumor volumes were about 375 mm3, and hypoxic percentages within tumors were measured by clonogenic excision assay procedures 72 h later. We found that partial hepatectomy increased the growth rates of the transplanted DLD-2 cancers by about a factor of 2.4. This increased growth rate was accompanied by an increase in the mitotic index from about 1.5 to 3.0%. Also, hypoxic fractions were decreased from 0.561 in control tumors to 0.235 in tumors from mice receiving partial hepatectomies. Unilateral nephrectomy and nonspecific surgery manipulations did not alter tumor growth rates, mitotic indices, or hypoxic percentages. These results indicate that partial hepatectomy produces significant alterations in tumor physiology. Results are consistent with a growth factor-mediated mechanism.     

Evidence for the functional binding in vivo of tumor rejection antigens to antigen-presenting cells in tumor-bearing hosts

Spleen cells of BALB/c mice bearing a syngeneic CSA1M fibrosarcoma were treated with anti-Thy-1.2 antibody plus C, yielding a T cell-depleted, APC-containing fraction. The APC-containing fraction was first tested for its capacity to present exogenous modified-self or another tumor (Meth A) Ag after in vitro pulsing. The results showed comparable Ag-presenting capacities to those obtained by APC-containing fraction from normal spleen cells, indicating that APC function is not affected in tumor-bearing mice. We next examined whether APC from CSA1M-bearing mice bind endogenously generated CSA1M tumor Ag onto its surfaces to stimulate tumor-specific T cells. Five rounds of inoculation of APC-containing fraction from CSA1M-bearing mice without further in vitro pulsing resulted in the induction of potent anti-CSA1M immune resistance. The involvement of anti-CSA1M T cells in the induction of anti-CSA1M immunity was excluded by the fact that the in vivo immunity was excluded by the fact that the in vivo immunity was delivered by Thy-1+ cell-depleted, but not by Thy-1+ cell-enriched fractions of spleen cells from CSA1M-bearing mice. Moreover, the failure of Sephadex G10-passed spleen cells to deliver anti-CSA1M resistance demonstrated the absolute requirement of APC for inducing the in vivo immunity. Finally, this in vivo resistance was found to be tumor specific, because APC fractions from CSA1M-bearing and Meth A-bearing BALB/c mice induced immune resistance selective against the corresponding tumor cell challenge. These results indicate that APC from tumor-bearing hosts can not only exert unaffected APC function against exogenous Ag, but also function to present tumor Ag generated endogenously in the tumor-bearing state and to produce tumor-specific immunity in vivo.     

Measurements of tumor tissue oxygen tension using a time-resolved luminescence-based optical oxylite probe: comparison with a paired survival assay

Recently, a system that measures tissue oxygen tension using time-resolved luminescence-based optical sensors has become available commercially (Oxford Optronix, Oxford, England). Two experiments were conducted using this system. First, the oxygen tension distribution was measured in two tumor lines: a spontaneous mouse fibrosarcoma, FSa-II, and a human squamous cell carcinoma xenograft, FaDu. The area in which the pO(2) was equal to or lower than 2.5 mmHg was defined as the hypoxic lesion, and the hypoxic cell fraction was taken as the fraction of these measurements in a tumor. The measured hypoxic cell fractions were compared with those determined by the paired cell survival assay for tumors of various sizes. Second, the tumor tissue pO(2) was measured continuously after administration of two different anesthetics to evaluate the effect of these drugs on tissue pO(2). Results indicated a good agreement between the hypoxic cell fractions measured by this system and those determined by the paired cell survival curve assay for tumors smaller than approximately 500 mm(3). For tumors larger than approximately 500 mm(3), the hypoxic cell fractions measured by the oxygen probe system were higher than those measured by the paired cell survival assay. This may suggest that the hypoxic cell fraction measured by the oxygen probes included both hypoxic and necrotic areas in large tumors where necrotic lesions occupied a significant portion of the tumor. Continuous measurements of pO(2) after anesthesia (Nembutal, or ketamine plus xylazine) showed a consistent rise in the pO(2) during the first 20-30 min of measurement. Subsequently, the pO(2) values became constant or continued to rise slowly. For comparison, the tumor cell survivals were assayed after a dose of 20 Gy given in air at 5, 20 and 60 min after anesthesia. The result showed a decrease in cell survival only in tumors irradiated 20 min after an injection of Nembutal.     

Hepatic zinc response via metallothionein induction after tumor transplantation

Based on previous findings that liver zinc and metallothionein (MT) levels increase after tumor transplantation, zinc metabolism in tumor-bearing mice was studied to clarify the role of zinc-MT in host defense systems. Zinc in the hepatic cytosolic MT fraction did not increase in tumor-bearing mice fed a zinc-deficient diet, suggesting that dietary zinc is necessary for apo-MT induction in the liver after tumor transplantation and is then incorporated into the apo-MT. When (65)ZnCl(2) was intravenously injected, liver (65)Zn levels in the tumor-bearing mice were higher than those in control mice for 72 h after the injection. Pancreatic and blood (65)Zn levels in tumor-bearing mice were lower than those in controls for 24 h (pancreas) and 6 h (blood) after the injection. These findings indicate that the hepatic zinc response via MT induction influences zinc metabolism in the body after tumor transplantation. Moreover, (65)Zn uptake in the liver of MT-deficient tumor-bearing mice was lower than that in control tumor-bearing mice 1 h after injection. (65)Zn uptake in the tumor and blood (65)Zn levels in the MT-deficient tumor-bearing mice were higher than those in the control tumor-bearing mice. Tumor weight increased more in MT-deficient mice than in control mice. The formation of zinc-MT in the liver of tumor-bearing mice might decrease blood zinc availability for tumors and other tissues, such as the pancreas.     

Multiple Molecular Forms of Dopamine β-Hydroxylase in the C1300 Mouse Neuroblastoma Tumor and in the Serum of Tumor-Bearing Mice

Abstract: The distribution of the enzymatic activity of dopamine β-hydroxylase (DBH) in linear sucrose gradients was studied for a soluble fraction of the C₁₃₀₀ mouse neuroblastoma tumor, for the serum of tumor-bearing A/J mice, and for adrenal tissue and serum of control mice. In controls (adrenal gland and serum of A/J mice), about 75% of the DBH activity was associated with a high-molecular-weight form, denoted as DBHA_A, with an apparent sedimentation coefficient of 11.3 S. About 25% of the DBH activity was attributable to a slower-sedimenting species (7.1 S), denoted as DBHB_B. In tumor supernatants and in the serum of tumor-bearing mice, about 55% of the DBH activity was present as the 7.1 S species (DBHR_B), while only 35% was recovered as the high-molecular-weight form (DBHA_A). Approximately 5% of the activity could be attributed to a separate form, with a sedimentation coefficient of about 4.5 S. This form is designated DBH_C. The ratio DBHR/DBHA is significantly higher in tumor tissue and in serum of tumor-bearing mice than in controls. The three enzymically active forms of DBH in the C₁₃₀₀ tumor are considered to represent the tetrameric (DBHA_C), dimeric (DBH_B), and monomeric (DBH_C) forms of the enzyme.     

Hypoxia Integration in the Serological Proteome Analysis Unmasks Tumor Antigens and Fosters the Identification of Anti-Phospho-eEF2 Antibodies as Potential Cancer Biomarkers

The expression by tumor cells of proteins with aberrant structure, expression or distribution accounts for the development of a humoral immune response. Autoantibodies (aAb) directed against tumor-associated antigens (TAA) may thus be particularly relevant for early detection of cancer. Serological proteome analysis (SERPA) aims to identify such circulating aAb through the immunoblotting of 2D-separated tumor cell proteins with cancer patient serum and the consecutive MS identification of proteins in reactive spots. This method has the advantage to use post-translationally modified proteins as a source of potential TAA. Here, we applied this strategy by using colorectal tumor cells pre-exposed to hypoxia in order to promote the expression of a pattern of TAA more likely to represent in vivo conditions. We used two human HCT116 and HT29 colorectal cancer cell lines exposed for 48 hours to 1% O₂. Spots positive after immunoblotting of 2D-separated lysates of hypoxic cells with the sera of tumor-bearing mice, were collected and analysed by MS for protein identification. Among the hypoxia-specific immunogenic proteins, we identified a phosphorylated form of eukaryotic translation elongation factor 2 (phospho-Thr56 eEF2). We confirmed the increased phosphorylation of this protein in hypoxic colorectal tumor cells as well as in mouse tumors. Using a specific immunoassay, we could detect the presence of corresponding anti-phospho-Thr56 eEF2 aAb in the serum of tumor-bearing mice (vs healthy mice). We further documented that the detection of these aAb preceded the detection of a palpable tumor mass in mice and validated the presence of anti-phospho-Thr56 eEF2 aAb in the serum of patients with adenomatous polyps and colorectal carcinoma. In conclusion, this study validates a phosphorylated form of eEF2 as a new TAA and more generally, provides evidence that integrating hypoxia upstream of SERPA offers a more relevant repertoire of TAA able to unmask the presence of circulating aAb.     

The influence of radiation dose on the magnitude and kinetics of reoxygenation in a C3H mammary carcinoma

The variation in hypoxic fraction as a function of time after various priming doses of radiation has been investigated in a C3H mouse mammary carcinoma in situ. The hypoxic fraction was calculated from data for local tumor control. Untreated tumors were found to contain 4.8% radiobiologically hypoxic cells. Within minutes after a priming dose of 20 Gy given in air, the hypoxic fraction increased to a value not significantly different from 100%. After 4 h, reoxygenation was complete (hypoxic fraction 1.3%), and the hypoxic fraction stabilized at a level significantly below the untreated value. Following a priming dose of 40 Gy the reoxygenation pattern was different: The hypoxic fraction stayed above the pretreatment value for 4 h, and pronounced reoxygenation occurred after 12 h (hypoxic fraction 0.4%). At longer time intervals the hypoxic fraction again increased to--and slightly above--the oxygenation level of untreated tumors. The present findings show that reoxygenation in solid tumors is a function of radiation dose, and the data suggest that mechanisms other than a decrease in tumor cell O2 consumption are involved in tumor reoxygenation.     

The change in hypoxic and chronically hypoxic cell fraction in murine tumors treated with hyperthermia

The effect of hyperthermia on the size of hypoxic and chronically hypoxic cell fractions in murine tumors was studied. The chronically hypoxic cell fraction was defined as a fraction of tumor cells which were not oxygenated under hyperbaric oxygen. Animals were C3Hf/Sed mice derived from our defined flora mouse colony. Tumors were FSa-II and MCa which were early generation isotransplants of a spontaneous fibrosarcoma and a mammary carcinoma, respectively. TCD50 (50% tumor control dose) or the radiation dose which yields a local tumor control in half the treated animals and TG (tumor growth) time or the time required for half the treated tumors to reach 1000 mm3 from the first treatment day were experimental end points. Hyperthermia was given by immersing animal feet into a water bath maintained at 43.5 +/- 0.1 degrees C. Animal tumors were irradiated with a 137Cs unit under hypoxic conditions, in air or under O2 30 psi. The hypoxic cell fraction increased immediately after hyperthermia in both MCa and FSa-II tumors. The chronically hypoxic cell fraction was, on the other hand, decreased following hyperthermia. The decrease was more substantial in the MCa than in FSa-II.     

Reversal of CD8+ T cell ignorance and induction of anti-tumor immunity by peptide-pulsed APC

In the present report, we have studied the potential of naive and activated effector CD8(+) T cells to function as anti-tumor T cells to a solid tumor using OVA-specific T cells from TCR-transgenic OT-I mice. Adoptive transfer of naive OT-I T cells into tumor-bearing syngeneic mice did not inhibit tumor cell growth. The adoptively transferred OT-I T cells did not proliferate in lymphoid tissue of tumor-bearing mice and were not anergized by the tumor. In contrast, adoptive transfer of preactivated OT-I CTL inhibited tumor growth in a dose-dependent manner, indicating that E.G7 was susceptible to immune effector cells. Importantly, naive OT-I T cells proliferated and elicited an anti-tumor response if they were adoptively transferred into normal or CD4-deficient mice that were then vaccinated with GM-CSF-induced bone marrow-derived OVA-pulsed APC. Collectively, these data indicate that even though naive tumor-specific T cells are present at a relatively high fraction they remain ignorant of the tumor and demonstrate that a CD8-mediated anti-tumor response can be induced by Ag-pulsed APC without CD4 T cell help.     

Z-100, a polysaccharide-rich preparation extracted from the human typeMycobacterium tuberculosis, improves the resistance of Meth-A tumor-bearing mice to endogenous septic infection

The effect of Z-100, an immunomodulatory arabinomannan extracted fromMycobacterium tuberculosis, on cecal ligation and puncture (CLP)-induced sepsis in mice bearing Meth-A fibrosarcoma was investigated. When normal BALB/c mice were subjected to the CLP procedure, their mortality rate was 17%. On the other hand, an increased mortality was observed in tumor-bearing mice subjected to CLP 10 days after tumor inoculation, and then all mice died when tumor- bearing mice were subjected to CLP 20 days after tumor inoculation. However, the increased percent mortality was decreased by 50% when these mice were injected intraperitoneally with a 10 mg/kg dose of Z-100. When splenocytes (5 × 10⁷ cells), obtained from Meth-A tumor-bearing mice 20 days after tumor inoculation, were transferred intravenously to normal mice (recipient mice), mortality of these recipient mice were increased by 62% as compared with that of the control (22%). However, no increased mortality (25%) was observed in recipient mice which were transferred with splenocytes from tumor-bearing mice injected intraperitoneally with Z-100 (10 mg/kg). In addition, suppressor cell activity was demonstrated in splenocytes from Meth-A tumor-bearing mice at 20 days after tumor inoculation using one-way mixed lymphocyte reaction. However, the suppressor cell activity was significantly decreased by the intraperitoneal administration of a 10 mg/kg dose of Z-100 (p<0.01). The increase of mortality in recipient mice by adoptive transfer of mononuclear cells (MNCs) from tumor-bearing mice was not detected when these MNCs were treated with anti-Thy 1.2 monoclonal antibody (mAb), anti-Lyt 2.2 mAb or anti-CD11b mAb, but an increase was seen with anti-Lyt 1.2 mAb or anti-immunoglobulin antiserum treated MNCs. These results suggest that the suppressor cells affect the mortality of CLP-induced sepsis and Z-100 may have a therapeutic activity against opportunistic infections in immunocompromised hosts through the regulation of suppressor T-cells.     

Characterization of suppressor cells in mice bearing syngeneic mastocytoma.

Suppressor cells from syngeneic P815 mastocytoma-bearing DBA/2 mice that inhibit in vitro generation of specific anti-tumor cytotoxicity were characterized. Suppressive activity was almost completely eliminated by treating suppressive spleen cells with anti-theta serum and complement. Treatment with anti-mouse lg serum and complement or with carbonyl iron did not affect their suppressive activity. When suppressive thymocytes from P815 tumor-bearing DBA/2 mice were tested for their capacity to inhibit the generation of cytotoxicity against L1210 cells, a leukemia line in DBa/2 mice, they did not affect the activity, indicating that the supressor cells in the thymocytes of P815 tumor-bearing mice are specific to the tumor. When Ficoll-Hypaque density cell separation was carried out with cytotoxic spleen cells and suppressive spleen cells from 815 tumor-bearing mice, the dense fraction was enriched for kiler cells whereas the suppressive activitty was mainly recovered in the light fraction. Therefore, killer cells and suppressor cells in P815 tumor-bearing mice are thought to be distinct populations.     

Analysis of electrophoretic mobility histograms of mouse splenocytes and thymocytes during tumor growth and after combined chemotherapy and immunotherapy

Changes in electrophoretic mobility histograms of splenocytes and thymocytes were studied in plasmacytoma X5563-bearing mice as an indicator of response to treatment with mitomycin C (MMC) alone or combined with the immunomodulator Krestin (PSK). Tumor growth was inhibited by 80-90% in the MMC-treated and was further inhibited in the MMC and PSK-treated group. Electrophoretic mobility histograms of splenocytes were used to determine the fraction of cells having intermediate mobility between high mobility (T cells) and low mobility (B cells). This fraction of intermediate-mobility cells increased in tumor-bearing mice, but a normal electrophoretic mobility pattern was obtained following successful antitumor treatment. In the electrophoretic mobility histogram of thymocytes, on the other hand, the low-mobility cells (cortical thymocytes) decreased in number during tumor growth and were further reduced in the MMC-treated group. This reduction was less in the MMC and PSK-treated group. These results suggest that combined therapy with MMC and PSK prevents damage of the host defence mechanism and allows more efficient antitumor treatment. Analysis of electrophoretic mobility histograms of splenocytes and thymocytes using a fully automated cell electrophoretic instrument makes possible the rapid evaluation of the immunological effects of drug therapy of tumor-bearing mice.     

In vivo anti-tumor immunity detected by leukocyte adherence inhibition

Summary The immune response of mice to a transplacentally induced alveolar cell tumor was studied with the leukocyte adherence inhibition (LAI) assay. The lung tumor, designated 85, was induced in a C3HfB/HeN (C3Hf) mouse by l-ethyl-l-nitrosourea (ENU). While a dose of 10⁵ cells of this tumor does not grow in syngeneic C3Hf mice, it does grow readily in (A×C3Hf)F₁ hybrid mice. The tumor possesses a tumor associated transplantation antigen (TATA) which cross-reacts with a normal tissue alloantigen in strain A/HeN (A) mice. Normal mice, tumor-immunized C3Hf mice, and tumor-bearing (A×C3Hf)F₁ mice possessed peritoneal cells, the majority of which adhered rapidly to glass and resisted gentle washing. When incubated with an extract of the 85 tumor, peritoneal cells from tumor-immunized mice demonstrated marked inhibition of adherence (62.4%) compared to similarly incubated peritoneal cells of either normal mice (30.3%) or tumor bearing mice (37.1%). Specificity of the reactivity in the LAI assay was demonstrated with a neuroblastoma extract and peritoneal cells from neuroblastoma-immunized C3Hf mice. Peritoneal cells from lung tumor-immunized mice, but not tumor-bearing mice, responded to a lung extract from strain A mice. In contrast to the microcytotoxicity assay, the LAI assay is capable of distinguishing the effective anti-tumor response of tumor-immunized C3Hf mice from the ineffective immune response of tumor-bearing (A×C3Hf)F₁ mice.     

Tumor accumulation of novel RES-avoiding liposomes.

For passive targeting of liposomes to tumor tissues, we earlier developed reticuloendothelial system (RES)-avoiding liposomes modified with a uronic acid derivative, palmityl-D-glucuronide (PGlcUA) (Namba, Y., Sakakibara, T., Masada, M., Ito, F. and Oku, N. (1990) Chem. Pharm. Bull. 38, 1663-1666). In this present study, we examined the blood clearance and biodistribution of PGlcUA-liposomes (dipalmitoylphosphatidylcholine/cholesterol/PGlcUA = 40:40:20 as a molar ratio) in normal and tumor-bearing mice. Liposomes containing dipalmitoylphosphatidylglycerol (DPPG) instead of PGlcUA was also examined as a control. When [3H]inulin-encapsulated PGlcUA-liposomes and DPPG-liposomes were intravenously injected into normal mice, approx. 50% of the 3H radioactivity was recovered from the liver, the bulk of RES, at 12 h after administration of DPPG-liposomes, while only approx. 20% of it was found there when PGlcUA-liposomes were administered. Radioactivity remaining in the plasma at 12 h after injection was 5-fold higher when PGlcUA-liposomes were injected than when DPPG-liposomes were used. Biodistribution of liposomes in tumor-bearing mice was also examined. Mice were inoculated with 10(7) S180 cells into the hind leg. After 1 week, liposomes were injected. Radioactivity of [3H]inulin originally encapsulated in the PGlcUA-liposomes accumulated in the tumor to an extent 3-4-fold higher than that of the marker in DPPG-liposomes. Liver/tumor ratio of the radioactivity was 12 for DPPG-liposomes and only 2 for PGlcUA-liposomes. This latter value is the lowest of various liposome formulations ever reported.     

Detection of different hypoxic cell subpopulations in human melanoma xenografts by pimonidazole immunohistochemistry

This study aimed at developing immunohistochemical assays for different subpopulations of hypoxic cells in tumors. BALB/c-nu/nu mice bearing A-07 or R-18 tumors were given a single dose of 90 mg/kg body weight or three doses (3 h apart) of 30 mg/kg body weight of pimonidazole hydrochloride intravenously. The fraction of pimonidazole-labeled cells was assessed in paraffin-embedded and frozen tumor sections and compared with the fraction of radiobiologically hypoxic cells. The staining pattern in paraffin-embedded sections indicated selective staining of chronically hypoxic cells. Frozen sections showed a staining pattern consistent with staining of both chronically and acutely/repetitively hypoxic cells. Fraction of pimonidazole-labeled cells in paraffin-embedded sections was lower than the fraction of radiobiologically hypoxic cells (single-dose and triple-dose experiment). In frozen sections, fraction of pimonidazole-labeled cells was similar to (single-dose experiment) or higher than (triple-dose experiment) fraction of radiobiologically hypoxic cells. Three different subpopulations of hypoxic cells could be quantified by pimonidazole immunohistochemistry: the fraction of cells that are hypoxic because of limitations in oxygen diffusion, the fraction of cells that are hypoxic simultaneously because of fluctuations in blood perfusion, and the fraction of cells that are exposed to one or more periods of hypoxia during their lifetime because of fluctuations in blood perfusion.     

In vivo activity of lymphokine-activated macrophages in host defense against neoplasia

Purified splenic macrophage (M phi) from normal DBA/2J mice and mice bearing P815 tumors were examined for responsiveness to lymphokine (LK) preparations containing high concentrations of IFN-gamma. For both normal and tumor-bearing M phi, LK treatment induced morphologic changes and increased the percentage of Ia+ cells from 35 to 55%. Although neither population exhibited spontaneous cytotoxicity toward P815 targets, LK treatment induced considerable tumoricidal activity in tumor-bearing M phi (32 to 80% lysis) but only minimal activity in normal M phi (8 to 17% lysis). Subcutaneous injection of 1 X 10(6)P815 cells into DBA/2J led to progressive tumor growth and death of 100% of the recipients after 27 +/- 3 days. Injection of a 1:18 mixture of P815 with either LK-activated normal or tumor-bearing M phi caused tumor regression after 10 days, and prolonged life until 43 +/- 4 days with tumor-bearing M phi and 39 +/- 3 days with normal M phi. Untreated normal or tumor-bearing M phi were unable to cause the effect (30 +/- 2 days), and lymphocytes could not be substituted for M phi (25 +/- 3 days). In x-irradiated recipients, no effect of LK-activated M phi could be observed (control = 19 +/- 2 days; LK-activated tumor-bearing M phi = 21 +/- 3 days). In addition, administration of an admixture of LK-treated M phi and x-rayed tumor before challenge with viable P815 enabled the recipient to inhibit tumor growth and caused tumor necrosis with inflammatory cell infiltration of the tumor. These observations suggest that, in part, LK-activated M phi may interact in vivo with host-derived cellular components and enhance the immune reactivity of the host against the tumor.     

Correlation of tumor oxygen dynamics with radiation response of the dunning prostate R3327-HI tumor

Our previous studies have shown that oxygen inhalation significantly reduces tumor hypoxia in the moderately well-differentiated HI subline of the Dunning prostate R3327 rat carcinoma. To test our hypothesis that modifying hypoxia could improve the radiosensitivity of these tumors, we performed experimental radiotherapy to compare the tumor response to ionizing radiation alone or in combination with oxygen inhalation. Tumor pO(2) measurements were performed on size-selected tumors several hours before radiotherapy using (19)F nuclear magnetic resonance echo planar imaging relaxometry (FREDOM) of the reporter molecule hexafluorobenzene. In common with our previous findings, the larger tumors (>3.5 cm(3)) exhibited greater hypoxia than the smaller tumors (<2 cm(3); P < 0.001), and oxygen inhalation reduced the hypoxic fraction (<10 Torr): In the larger tumors, hypoxic fraction dropped significantly from a mean baseline value of 80% to 17% (P < 0.001). The effect of oxygen administered 30 min before and during irradiation on tumor response to a single 30-Gy dose of photons was evaluated by growth delay. For the smaller tumors, no difference in growth delay was found when treatment was given with or without oxygen breathing. By contrast, breathing oxygen before and during irradiation significantly enhanced the growth delay in the larger tumors (additional 51 days). The differential behavior may be attributed to the low baseline hypoxic fraction (<10 Torr) in small tumors (20%) as a target for oxygen inhalation. There was a strong correlation between the estimated initial pO(2) value and the radiation-induced tumor growth delay (R > 0.8). Our histological studies showed a good match between the perfused vessels marked by Hoechst 33342 dye and the total vessels immunostained by anti-CD31 and indicated extensive perfusion in this tumor line. In summary, the present results suggest that the ability to detect modulation of tumor pO(2), in particular, the residual hypoxic fraction, with respect to an intervention, could have prognostic value for predicting the efficacy of radiotherapy.     

Eradication of tumor cells after injection into immunized hosts compared with the eradication of tumor cells after transfer of immune peritoneal exudates into tumor-bearing recipients

Summary DBA/2 mice were immunized against the syngeneic SL2 lymphoma by two or five injections with irradiated lymphoma cells given IP or SC, respectively. The antitumor efficacy induced in immunized mice was tested by (a) IP injection of the immunized mice with nonirradiated tumor cells and (b) transfer of the total immune peritoneal exudate, the cellular fraction only, or the cell-free fraction only, IP into tumor-bearing recipients, or (c) tumor neutralization tests (Winn assay). It was shown that immunized mice were able to reject 5×10⁷ SL2 tumor cells (8 of 14 mice survived >100 days), while in most transfer experiments 2×10⁵ SL2 cells could be eradicated. In the tumor neutralization experiments a number of 10⁶ SL2 cells were eradicated. When the immune exudates were given before the inoculations of SL2 tumor cells the number of survivors increased significantly. Further, it was shown that the cellular fraction is the major contributor to the antitumor effect in the transfer experiments, since there was no significant difference in tumor eradication after injection of a complete immune exudate and after injection of the isolated cellular fraction. Injection of the noncellular fraction had no measurable antitumor effect. An increase in the number of injections with total peritoneal exudates from immunized mice did not result in an increase in tumor eradication in the tumor-bearing recipients.Extra stimulation (IP) of immunized mice with 10⁴ nonirradiated cells 6 days after the last immunization resulted in an increase of the antitumor efficacy of these peritoneal exudates of these mice when collected 4–24 h after this stimulation. Extra stimulation with 10⁶ irradiated cells had no measurable effect.     

Tumor-bearing mice exhibit a progressive increase in tumor antigen-presenting cell function and a reciprocal decrease in tumor antigen-responsive CD4+ T cell activity.

Splenic CD4+ T cells from BALB/c mice bearing a syngeneic tumor (CSA1M) 2 to 3 wk after the inoculation with CSA1M cells produced IL-2 and macrophage-activating factor upon in vitro cultures. This lymphokine production was achieved without stimulation of these T cells with exogenous stimulating tumor Ag. However, elimination of APC from spleen cells resulted in almost complete abrogation of the capacity of CD4+ T cells to produce IL-2/macrophage-activating factor. The lymphokine production was regained when APC from CSA1M-bearing mice were added back to cultures. APC from normal or another syngeneic tumor (Meth A)-bearing mice failed to regain the lymphokine production. These observations demonstrated that the lymphokines were produced by CD4+ T cells from CSA1M-bearing hosts through their collaboration with APC binding CSA1M tumor Ag in the tumor-bearing state. The lymphokine-producing capacity of whole spleen cells from tumor-bearing mice reached the maximal level around 2 to 3 wk after tumor implantation but gradually decreased with the progress of tumor-bearing stages. Importantly, tumor-bearing stage-related changes were observed in a different fashion in the capacities of anti-CSA1M CD4+ T cells vs CSA1M tumor Ag-binding APC. The capacity of APC increased with the progress of tumor-bearing stages as demonstrated by the stimulation of CSA1M-immunized T cells with APC from different CSA1M-bearing stages. In contrast, the reactivity of anti-CSA1M T cells to APC from a given CSA1M-bearing stage decreased with the tumor-bearing stage. These results demonstrate a stage-related increase tumor Ag-binding APC function, as well as a reciprocal reduction in tumor Ag-responsive CD4+ T cell activity.