Mouse Tumor Systems Research Articles

Tumor-derived thymic stromal lymphopoietin enhances lung metastasis through an alveolar macrophage-dependent mechanism

ABSTRACTIt is well-recognized that macrophages, which arise from circulating precursors, enhance tumor progression in patients and animal models. However, less is known regarding the role of tissue-resident macrophages in metastasis. Moreover, the identification of tumor factors which influence macrophage function in the metastatic niche remains incomplete. Here, we investigated one such cytokine known as thymic stromal lymphopoietin (TSLP). Our rationale to focus on TSLP was based on two non-overlapping findings; first, TSLP exacerbates asthma in part by altering the lung macrophage response and, secondly, TSLP is produced by certain mouse and human tumor systems, although its role in neoplasia remains understudied. Thus, we tested the hypothesis that tumor-derived TSLP augments lung metastasis by rendering alveolar macrophages pro-tumorigenic. To test this hypothesis, we principally employed the 4T1 tumor model, which produces high levels of TSLP and metastasizes to the lung. TSLP loss-of-function significantly reduced spontaneous lung metastasis, as well as lung colonization. Moreover, similar outcomes were observed in both wild-type and immune-deficient hosts, suggesting that TSLP acted on innate immune cells such as macrophages. To test this notion, pharmacologic depletion of alveolar macrophages significantly reduced lung tumor growth of the TSLP-expressing, but not TSLP-deficient tumor population. In contrast, depleting macrophages originating from the circulation did not impact lung tumor growth. Lastly, TSLP increased the invasive and angiogenic gene expression profile of the alveolar macrophage population. Altogether, our study identified a novel TSLP-alveolar macrophage axis in lung metastasis, which offers new insights into mechanisms of metastasis and potential therapeutic targets.

Soluble CD80 Protein Delays Tumor Growth and Promotes Tumor-Infiltrating Lymphocytes.

Tumor cells use various immune-suppressive strategies to overcome antitumor immunity. One such method is tumor expression of programmed death ligand-1 (PD-L1), which triggers apoptotic death or anergy upon binding programmed death-1 (PD-1) on T cells. Our previous in vitro cellular studies with human and mouse PD-L1+ tumor cells demonstrated that a soluble form of the costimulatory molecule CD80 prevented PD-L1-mediated immune suppression and restored T-cell activation by binding PD-L1 and blocking interaction with PD-1. We now report that in vivo treatment of established syngeneic PD-L1+ CT26 colon carcinoma and B16F10 melanoma tumors with CD80-Fc delays tumor growth and promotes tumor-infiltrating T cells. Studies with PD-1-/- and CD28-/- mice demonstrate that soluble CD80 acts in vivo by simultaneously neutralizing PD-1 suppression and activating through CD28. We also report that soluble CD80 mediates its effects by activating transcription factors EGR1-4, NF-κB, and MAPK, downstream signaling components of the CD28 and T-cell receptor pathways. Soluble CD80 binds to CTLA-4 on activated human peripheral blood mononuclear cells. However, increasing quantities of CTLA-4 antagonist antibodies do not increase T-cell activation. These results indicate that soluble CD80 does not suppress T-cell function through CTLA-4 and suggest that CTLA-4 acts as a decoy receptor for CD80, rather than functioning as a suppressive signaling receptor. Collectively, these studies demonstrate that soluble CD80 has therapeutic efficacy in vivo in mouse tumor systems and that its effects are due to its ability to inhibit PD-1-mediated suppression while concurrently activating T cells through CD28. Cancer Immunol Res; 6(1); 59-68. ©2017 AACR.

Cryotherapy with concurrent CpG oligonucleotide treatment controls local tumor recurrence and modulates HER2/neu immunity.

Percutaneous cryoablation is a minimally invasive procedure for tumor destruction, which can potentially initiate or amplify antitumor immunity through the release of tumor-associated antigens. However, clinically efficacious immunity is lacking and regional recurrences are a limiting factor relative to surgical excision. To understand the mechanism of immune activation by cryoablation, comprehensive analyses of innate immunity and HER2/neu humoral and cellular immunity following cryoablation with or without peritumoral CpG injection were conducted using two HER2/neu(+) tumor systems in wild-type (WT), neu-tolerant, and SCID mice. Cryoablation of neu(+) TUBO tumor in BALB/c mice resulted in systemic immune priming, but not in neu-tolerant BALB NeuT mice. Cryoablation of human HER2(+) D2F2/E2 tumor enabled the functionality of tumor-induced immunity, but secondary tumors were refractory to antitumor immunity if rechallenge occurred during the resolution phase of the cryoablated tumor. A step-wise increase in local recurrence was observed in WT, neu-tolerant, and SCID mice, indicating a role of adaptive immunity in controlling residual tumor foci. Importantly, local recurrences were eliminated or greatly reduced in WT, neu tolerant, and SCID mice when CpG was incorporated in the cryoablation regimen, showing significant local control by innate immunity. For long-term protection, however, adaptive immunity was required because most SCID mice eventually succumbed to local tumor recurrence even with combined cryoablation and CpG treatment. This improved understanding of the mechanisms by which cryoablation affects innate and adaptive immunity will help guide appropriate combination of therapeutic interventions to improve treatment outcomes.

Lack of in vivo cross-resistance with 4′-thio-ara-C against drug-resistant murine P388 and L1210 leukemias

4'-Thio-β-D-arabinofuranosylcytosine (4'-thio-ara-C), which has shown a broad spectrum of antitumor activity against human tumor systems in mice and is undergoing clinical trials, was evaluated for cross-resistance to seven clinical agents in order to identify potentially useful guides for patient selection for further clinical trials of 4'-thio-ara-C and possible noncross-resistant drug combinations with 4'-thio-ara-C. A drug resistance profile for 4'-thio-ara-C, which was administered intraperitoneally daily for nine consecutive days, was obtained using seven drug-resistant P388 and L1210 leukemias that were implanted intraperitoneally in mice. Multidrug-resistant P388 leukemias (leukemias resistant to doxorubicin, etoposide, or paclitaxel) exhibited no cross-resistance to 4'-thio-ara-C. Leukemias resistant to camptothecin, cisplatin, and 5-fluorouracil were also not cross-resistant to 4'-thio-ara-C. Only the leukemia resistant to 1-β-D-arabinofuranosylcytosine was cross-resistant to 4'-thio-ara-C. The data suggest that (1) it may be important to exclude or to monitor with extra care patients who have previously been treated with 1-β-D-arabinofuranosylcytosine and (2) the lack of cross-resistance seen with 4'-thio-ara-C may contribute to therapeutic synergism when 4'-thio-ara-C is combined with other agents.

Chemotherapy Induces Tumor Clearance Independent of Apoptosis

Dysregulation of apoptosis is associated with the development of human cancer and resistance to anticancer therapy. The ultimate goal of cancer treatment is to selectively induce cancer cell death and overcome drug resistance. A deeper understanding of how a given chemotherapy affects tumor cell death is needed to develop strategically designed anticancer agents. Here, we use a xenograft mouse tumor system generated from genetically defined cells deficient in apoptosis to examine the involvement of multiple forms of cell death induced by cyclophosphamide (CP), a DNA alkylating agent commonly used in chemotherapy. We find that although apoptosis facilitates tumor regression, it is dispensable for complete tumor regression as other forms of cell death are activated. Sporadic necrosis is observed in both apoptosis-competent and deficient tumors evident by tumor cell morphology, extracellular release of high mobility group box 1 protein, and activation of innate immune cells in CP-treated tumors. Our findings indicate that in apoptosis-deficient tumors, necrosis may play a fundamental role in tumor clearance by stimulating the innate immune response.

Systemic Administration of IL-23 Induces Potent Antitumor Immunity Primarily Mediated through Th1-Type Response in Association with the Endogenously Expressed IL-12

IL-23, a cytokine, which is composed of the p40 subunit shared with IL-12 and the IL-23-specific p19 subunit, has been shown to preferentially act on Th1 effector/memory CD4+ T cells and to induce their proliferation and IFN-gamma production. The IL-23 is also reported to act on Th17-CD4+ T cells, which are involved in inducing tissue injury. In this study, we examined the antitumor effects associated with systemic administration of IL-23 and their mechanisms in mouse tumor system. Systemic administration of high-dose IL-23 was achieved using in vivo electroporation of IL-23 plasmid DNA into the pretibial muscles of C57BL/6 mice. The IL-23 treatment was associated with significant suppression of the growth of pre-existing MCA205 fibrosarcoma and prolongation of the survival of treated mice without significant toxicity when compared with those of the mice treated with EGFP. Although the therapeutic outcomes were similar to those with the IL-12 treatment, the IL-23 treatment induced characteristic immune responses distinctive to those of IL-12 treatment. The IL-23 administration even at the therapeutic levels did not induce detectable IFN-gamma concentration in the serum. In vivo depletion of CD4+ T cells, CD8+ T cells, or NK cells significantly inhibited the antitumor effects of IL-23. Furthermore, the CD4+ T cells in the lymph nodes in the IL-23-treated mice showed significant IFN-gamma and IL-17 response upon anti-CD3 mAb stimulation in vitro. These results and the ones in the IFN-gamma or IL-12 gene knockout mice suggest that potent antitumor effects of IL-23 treatment could be achieved when the Th1-type response is fully promoted in the presence of endogenously expressed IL-12.

Preclinical antitumor activity of 4'-thio-beta-D-arabinofuranosylcytosine (4'-thio-ara-C).

4'-Thio-beta -d-arabinofuranosylcytosine (4'-thio-ara-C), which has shown significant cytotoxicity against a panel of human tumor lines, was evaluated for antitumor activity against a spectrum of human tumor systems in mice. Antitumor activity was evaluated in 15 subcutaneously implanted human tumor xenografts. 4'-Thio-ara-C was administered intraperitoneally using either q1dx9 (daily treatment for nine consecutive days) or q4hx3/q1dx9 (three treatments each day separated by 4-h intervals for nine consecutive days). 4'-Thio-ara-C exhibited an excellent spectrum of activity. Treatment with the compound was curative against HCT-116 colon, SW-620 colon, NCI-H23 NSCL, and CAKI-1 renal tumors and resulted in partial/complete regressions in the DLD-1 colon, NCI-H522 NSCL, DU-145 prostate, and PANC-1 pancreatic tumor models. Tumor stasis was noted for HT29 colon and NCI-H460 NSCL tumors. Tumor inhibition was observed for A549 NSCL, PC-3 prostate, LNCAP prostate, and MDA-MB-435 breast tumors. Of the 15 tumors examined, only CFPAC-1 pancreatic was unresponsive to the compound. In contrast, 1-beta -d-arabinofuranosylcytosine was minimally active at best against CAKI-1 renal, HCT-116 colon, NCI-H460 NSCL, and SW-620 colon tumors. Schedule- and route-dependency studies were conducted using the NCI-H460 NSCL tumor. The activity of 4'-thio-ara-C was independent of schedule when comparing q2dx5 (every other day for five treatments), q1dx9, and q4hx3/q1dx9 treatment schedules. 4'-Thio-ara-C was equally effective by the intravenous and intraperitoneal routes of administration, with the oral route being less efficacious. On the basis of these results, 4'-thio-ara-C appears to have a profile distinct from other nucleoside antitumor agents and is being advanced to clinical trials.

Current status of knowledge and critical issues in tumor oxygenation. Results from 25 years research in tumor pathophysiology.

In 1972 we started the systematic investigation of oxygenation status in (a) experimental tumor systems in rats and mice [e.g., 1–6], (b) in human tumors xenotransplanted into immune-deficient rnu/rnu-rats [e.g., 7–9], (c) in tumors derived from spontaneously tumorigenic or ras-transformed cell lines [10], and (d) in human tumors in situ [e.g., 11–15] under various pathophysiological conditions using polarographic O2 needle electrodes. In the earlier studies a cryospectrophotometric ex vivomicrotechnique was also used that allowed the measurement of HbO2 saturation in individual red blood cells in tumor microvessels [e.g., 16, 17]. Besides polarographic and cryospectrophotometric microtechniques, mathematical evaluations of the pO2 distribution in tumor tissues have additionally been used to gain an insight into the oxygenation of microareas in tumor tissues [e.g., 18–21].KeywordsMetastatic LesionUterine CervixOxygenation StatusTumor HypoxiaTumor OxygenationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Pretargeting

BACKGROUND. Human imaging studies show that maximum human tumor concentrations of monoclonal antibody (MoAb) are achieved in 1 day, but several days are required for background reduction and sensitive radioimmunoscintigraphy of tumors. With therapeutic radionuclides such as yttrium-90 ( 90 Y), this long biologic half-life imposes a high radiation burden on sensitive normal tissues from the large amount of unlocalized radioactivity. Normal tissue toxicity, especially to the bone marrow, has been the major limiting factor in the application of radioimmunotherapy to solid tumors. Pretargeting techniques provide an alternative method with which to obtain high selective tumor uptake of 90 Y with simultaneous minimization of nontarget tissue background. METHODS. Current MoAb techniques employ either the biotin/avidin or the hapten/antibody system. DNA/DNA and prodrug/enzyme systems also have been used and many other ligand/receptor systems are possible. All the methods depend on a long circulating conjugate to obtain high target uptake with a diffusible, rapidly excreted effector molecule. RESULTS. Fast in, slow out tumor kinetics, ideal for therapy, have been achieved. In one mouse tumor system the biologic half-life of a bivalent 88 Y JANUS tetraazacyclodidecanetetraacetic acid hapten, measured over 5 days, was approximately 24 hours. The therapeutic ratio, obtained from the integrated tumor and blood concentrations over 5 days, was approximately 20/1 compared with 2-3/1 with directly labeled MoAb. The total injected dose remaining in the mouse at 24 hours was 5.5%, 23% of which was in the tumor. CONCLUSIONS. These result suggest that it may be possible to deliver tumoricidal radiation doses with 90 Y using pretargeting techniques without severe normal bone marrow irradiation.

Pretargeting: general principles; October 10-12, 1996.

Human imaging studies show that maximum human tumor concentrations of monoclonal antibody (MoAb) are achieved in 1 day, but several days are required for background reduction and sensitive radioimmunoscintigraphy of tumors. With therapeutic radionuclides such as yttrium-90 (90Y), this long biologic half-life imposes a high radiation burden on sensitive normal tissues from the large amount of unlocalized radioactivity. Normal tissue toxicity, especially to the bone marrow, has been the major limiting factor in the application of radioimmunotherapy to solid tumors. Pretargeting techniques provide an alternative method with which to obtain high selective tumor uptake of 90Y with simultaneous minimization of nontarget tissue background. Current MoAb techniques employ either the biotin/avidin or the hapten/antibody system. DNA/DNA and prodrug/enzyme systems also have been used and many other ligand/receptor systems are possible. All the methods depend on a long circulating conjugate to obtain high target uptake with a diffusible, rapidly excreted effector molecule. Fast in, slow out tumor kinetics, ideal for therapy, have been achieved. In one mouse tumor system the biologic half-life of a bivalent 88Y JANUS tetraazacyclodidecanetetraacetic acid hapten, measured over 5 days, was approximately 24 hours. The therapeutic ratio, obtained from the integrated tumor and blood concentrations over 5 days, was approximately 20/1 compared with 2-3/1 with directly labeled MoAb. The total injected dose remaining in the mouse at 24 hours was 5.5%, 23% of which was in the tumor. These result suggest that it may be possible to deliver tumoricidal radiation doses with 90Y using pretargeting techniques without severe normal bone marrow irradiation.

New antitumor substances, FR901463, FR901464 and FR901465. II. Activities against experimental tumors in mice and mechanism of action.

FR901463, FR901464 and FR901465, novel antitumor substances, were isolated from the fermentation broth of Pseudomonas sp. No. 2663. Their antitumor activities were examined in three mouse tumor systems and one human tumor system. The three FR compounds prolonged the life of mice bearing murine ascitic tumor P388 leukemia (T/C values were 160%, 145% and 127% for FR901463, FR901464 and FR901465, respectively), and inhibited the growth of a human solid tumor, A549 lung adenocarcinoma, with different effective dose ranges. FR901464 exhibited most prominent effects on these tumor systems among the three FR compounds. FR901464 also inhibited the growth of murine solid tumors, Colon 38 carcinoma and Meth A fibrosarcoma. To address the involvement of transcriptional activation ability of the three FR compounds in the antitumor effect, we selected FR901464 as a candidate compound and investigated cell cycle transition, chromatin status and endogenous gene expression in FR901464-treated tumor cells having elevated transcriptional activity. FR901464 induced characteristic G1 and G2/M phase arrest in the cell cycle and internucleosomal degradation of genomic DNA with the same kinetics as activation of SV40 promoter-dependent cellular transcription in M-8 tumor cells. In contrast to the potent activation of the viral promoter, FR901464 suppressed the transcription of some inducible endogenous genes but not house keeping genes in M-8 cells. These results suggest that FR901464 may induce a dynamic change of chromatin structure, giving rise to strong antitumor activity, and therefore may represent a new type of drug for cancer chemotherapy.

Bacteriochlorophyll- a as photosensitizer for photodynamic treatment of transplantable murine tumors

Bacteriochlorophyll- a (bChl a), which absorbs light of 780 nm wavelength, was tested for in vivo photodynamic activity in the SMT-F and RIF transplantable mouse tumor systems. High performance liquid chromatography (HPLC) analysis of tissue extracts showed that bChl a was rapidly degraded in vivo to bacteriopheophytin- a (bPheo a) and other breakdown products. These were also photodynamically active, and tumor response could be achieved over a wavelength range of 660 to 780 nm, while tumor cure was restricted to wavelengths of 755 (bPheo a) to 780 nm. A photosensitizing product absorbing at 660 nm was also present in isolated tumor cells. Photodynamic cell kill of tumor cells isolated from tumors after bChl a accumulation in vivo, using 755 or 780 nm light vitro, was exponential up to 20–40 J cm −2. Above this light dose little or no further damage could be achieved, which is an indication of the rapid photobleaching of these sensitizers. In vivo, vascular occlusion occurred readily if light treatment was delivered shortly after sensitizer administration, but was delayed if light treatment was carried out 24 h after injection. Although up to 70% of tumor cells were lethally damaged after completion of in vivo light treatment, concurrent severe vascular destruction seemed necessary for tumor cure. Normal tissue photosensitivity totally subsided within 5 days after sensitizer administration.

Combination therapy of pions and SPG (Sonifilan, Schizophyllan), a biological response modifier for mouse tumor systems

Female C3H mice aged 8–10 weeks with transplanted KHT sarcoma or SCCVII tumor were used to investigate the antitumor effect of SPG (Sonifilan, Schizophyllan) alone and in combination with local irradiation of pions with 4 fractions of 400 cGy (total 1600 cGy). Daily doses of 10 mg/kg of SPG were given intramuscularly to the mice bearing KHT sarcoma for 14 consecutive days from day 7, and to mice bearing SCCVII tumor for 20 consecutive days from day 7 and thereafter three times a week for another 2 weeks. The antitumor effect was evaluated by the changes in tumor volume, survival curves, and the number of pulmonary metastatic nodules on the surface of the lungs. SPG failed to exert any antitumor effect and any life-prolonging effect for the KHT sarcoma. As for SCCVII tumor, in the group treated with pions and SPG, tumor growth decreased significantly ( p < 0.01) compared with the group treated with pion only, and life prolonging effect and metastasis-suppressing effect were also observed ( p < 0.05). In conditions of minimal residual disease brought about by pion irradiation, the adjuvant effect of a Biological Response Modifier (BRM) SPG may prove to be a promising method of cancer therapy for some tumors.

Effects of dietary fat and protein on DMH‐induced tumor development and immune responses

Although in three different mouse tumor systems with corn oil as dietary fat we previously found that milk protein decreased tumor development compared with beef, the results were reversed in 1,2-dimethylhydrazine (DMH)-injected mice. The purpose of this study was to determine if the latter result was due to the protein source. BALB/c mice (n = 280) were divided into five diet groups and injected 10 times at weekly intervals with DMH (20 mg/kg wt) or saline. Four diets contained 11% protein (casein, milk, or beef) and 5% fat (corn oil or beef tallow), and the AIN-76A diet was used as a control diet. The source of fat was a significant modulator of tumor development. Corn oil markedly increased total tumor volume and the number of tumors per mouse compared with beef tallow. Its tumor-enhancing effects were evident when it was combined with milk but not with casein. In addition, significantly lower lymphoproliferation and T-cell cytotoxicity against colon tumor cell targets was associated with corn oil consumption, whereas nonfat milk as the protein source was related to normal oxidative burst capacity of phagocytes. These results demonstrate that the source of dietary fat, in addition to the protein source, has a profound effect on both tumor development and immune responsiveness in this animal tumor system.

Chlorpromazine-Induced Hypothermia in Tumour-Bearing Mice, Acute Cytotoxic Drug Lethality and Long-Term Survival

The acute lethality of various cytotoxic drugs (doxorubicin, vinblastine and nitrogen mustard) and long-term survival in a syngenic mouse-tumour system were studied at normal body temperature and at 28 degrees C induced by chlorpromazine. Chlorpromazine-induced hypothermia itself neither caused acute toxicity nor influenced long-term survival. Doxorubicin (15 mg/kg) and nitrogen mustard (6 mg/kg) lethality was reduced at decreased temperature. The median survival time increased significantly from 35 days in normothermic to 52 days in hypothermic doxorubicin-treated mice. With nitrogen mustard, no increase in long-term survival was seen in the hypothermic group. The acute lethality of vinblastine was enhanced by hypothermia and the long-term tumour survival was unaffected. Hypothermia or possibly chlorpromazine considerably modulates drug toxicity and possibly anti-tumoural activity.

The influence of the surgical wound on local tumor recurrence.

Failure of a primary surgical treatment for cancer is often caused by recurrence of the tumor at the surgical site. The KHT mouse tumor system recapitulates this experience and provides a useful model to test strategies for reducing the incidence of local recurrence after surgical excision. There was an 82% local recurrence of the KHT tumor after surgery. A cell dilution assay indicated that it would require only 39 tumor cells injected into the wound site to result in the same (82%) incidence of tumors. This figure is in contrast to 340 cells required when the cells were injected into an unwounded flank. With the B16 melanoma in C57B1 mice and the Meth A sarcoma in BALB/c mice, the number of cells necessary to induce a tumor (TD/50) was also significantly reduced when the cells were injected into a surgical wound rather than into nonwounded tissue. The difference in cell number was interpreted as the result of the presence of growth factors derived from the traumatized tissue and the inflammatory cells at the wound site. Neither a 5 nor a 15 Gy dose of x-radiation delivered to the wound site immediately after surgical excision of the KHT tumor resulted in a significant reduction in the incidence of local recurrences. When the same doses of x-radiation were given immediately after injecting 36 KHT cells into a wound, no tumors developed. This difference was believed to have resulted from the hypoxic condition in the wound site and the presence of residual clonogenic tumor cells in a nonproliferating (radioresistant) state.

A new antitumor antibiotic, FR900840. III. Antitumor activity against experimental tumors.

FR900840 [2S)-2-amino-2-carboxyethyl (3R)-2-diazo-3-hydroxybutyrate), a new antibiotic with antitumor activity was isolated from the fermentation broth of Streptomyces sp. No. 8727. Its antitumor activity was examined in three mouse tumor systems and ten human tumor systems. FR900840 had no clear effect on mouse ascitic tumors, P388 and L1210, and the B16 melanoma line, but had prominent antitumor effects on several human solid tumors. Its antitumor activity against A549 human lung adenocarcinoma was stronger than those of vinblastine, doxorubicin and cisplatin. These results suggest that FR900840 may become a useful prototype antitumor drug.

The theory of photodynamic therapy dosimetry: consequences of photo-destruction of sensitizer.

Photodynamic dose is defined as the area under the curve of sensitizer level plotted as a function of light dose. This is a photochemical definition of dose. We will show that this definition is useful in predicting photobiological response. The photodestruction of sensitizer during photodynamic therapy is shown to result in an upper limit on the photodynamic dose which can be delivered by an unlimited light dose. This limit results in the opportunity to make total photodynamic dose uniform to considerable depths (one to two centimeters). The existence of thresholds for permanent tissue damage allows protection of normal tissue from the large light doses required to achieve this limiting dose deep in the tissue. Higher sensitizer levels in the tumor permit tumor destruction while the normal tissues are protected. A clinical trial to determine the proper level of injected dose necessary for these results is required. This theory of photodynamic therapy (PDT) dosimetry is tested in the DBA-SMT experimental mouse tumor system. Combinations of drug and light which are not reciprocal but are nearly equal by this theory are shown to give equivalent tumor control at seven days post treatment. Reciprocal combinations of drug and light fail to give equivalent results when they ae selected using the theory to choose a combination where reciprocity should fail.

Energy Metabolism and Blood Perfusion in a Mouse Mammary Adenocarcinoma during Growth and Following X Irradiation

Biochemical and blood perfusion changes in a mouse tumor system (MDAH MCaIV) were studied relative to normal tissues under conditions of normal blood flow and clamped blood supply. Further studies were performed during tumor growth and after local X irradiation. The biochemical profiles of three untreated human soft tissue sarcomas were also investigated. Animal tumors were irradiated in situ with either a single or fractionated regime to total doses of 20 or 49 Gy. Assays of lactate, pyruvate, AMP, ADP, and ATP were made on freeze-clamped tissue following authentic or sham treatments. Blood perfusion to tumors treated in the same way was measured using iv injection of 201Tl. The human tumors were found to have a lower lactate to pyruvate ratio (L/P) than the MCaIV tumors; their ATP levels were also lower. L/P was much higher in the MCaIV tumors than in normal liver, kidney, and muscle in the mouse. Occlusion of the blood supplies of the normal kidney and the MCaIV tumor caused an increase in the lactate and L/P levels in both cases. However, whereas the ATP level in the kidney fell, the level in the tumor was maintained. There was some evidence that the adenine nucleotides were not in equilibrium via the adenyl kinase catalyzed reaction. In addition, tumors were found to contain the enzyme creatine kinase. These results suggest that energy charge calculations cannot be computed in a meaningful manner because the creatine kinase catalyzed phosphorylation of ADP would maintain a higher than normal ATP level. Lactate and L/P ratio was found to increase during tumor growth and decrease following X irradiation. The total adenine nucleotides (AMP + ADP + ATP) exhibited a trend toward lower values with increasing tumor size. There was no significant change in total adenine nucleotides after a single 20-Gy dose; however, fractionated radiation caused some fall in total nucleotides. It is concluded that, in this tumor system, lactate level is a sensitive index of radiation-induced biochemical changes which are likely to reflect changes in tumor oxygenation.

Generation of lymphokine-activated killer (LAK) cells from tumor-infiltrating lymphocytes

Culture of tumor-infiltrating lymphocytes (TIL) containing about 20% BMC2 tumor cells with recombinant human interleukin 2 (rIL-2) resulted in the diminish of tumor cells and the growth of lymphocytes. These IL-2-activated lymphocytes showed a strong cytotoxic activity against not only syngeneic tumor cells but also allogeneic tumor cells. Such broad-reactive killer cells, termed lymphokine-activated killer (LAK) cells, are also inducible from spleen cells by in vitro activation with IL-2. However, LAK cells generated from TIL (TIL-LAK) showed higher cytotoxic activity against BMC2 than LAK cells generated from spleen cells (S-LAK). Furthermore, it was demonstrated that TIL-LAK cells revealed marginal cytotoxic activity against normal Con A blasts and YAC-1 cells as opposed to S-LAK. Flow cytometric analysis of TIL-LAK indicated that TIL-LAK cells mainly consisted of Thy 1.2 +, Ly 2 +, asialo GM1 + cells. TIL-LAK cells displayed not only in vitro cytotoxicity but also in vivo anti-tumor activity. Furthermore, it was also confirmed that TIL-LAK cells could be induced in autochthonous mouse tumor systems and human gastric tumor systems.