Insulin-tumour interrelationship in EL4 lymphoma or thymoma-bearing mice. I. Alloxan-diabetic or non-diabetic mice

A study has been carried out in which a comparison was made between EL4 lymphoma (assumed to be an insulin-producing secreting tumour) and thymoma (an insulin-dependent tumour). Tumour development and incidence, 3H-thymidine incorporation and insulin content in tumours, the host's food intake, blood insulin, glucose and cholesterol were determined in non-diabetic and alloxan-diabetic mice. Whereas no significant differences were observed between the diabetic and non-diabetic EL4 tumour-bearing mice, the diabetic, thymoma tumour-bearing mice showed reduced tumour growth and lower tumour incidence as compared with their non-diabetic counterparts. Insulin administration to diabetic tumour bearing mice, enhanced 3H-thymidine incorporation in the thymoma tumour cells only, and the insulin content of the EL4 tumours was found to be higher than that of the thymoma tumours. Rapid diabetes remission was observed in the diabetic, EL4 tumour-bearing mice as compared with the thymoma tumour-bearing mice.     

Gadd45a acts as a modifier locus for lymphoblastic lymphoma.

GADD:45a-/- and p53-/- mice and cells derived from them share similar phenotypes, most notably genomic instability. However, p53-/- mice rapidly develop a variety of neoplasms, while Gadd45a-/- mice do not. The two proteins are involved in a regulatory feedback loop, whereby each can increase the expression or activity of the other, suggesting that common phenotypes might result from similar molecular mechanisms. Mice lacking both genes were generated to address this issue. Gadd45a-/-p53-/- mice developed tumors with a latency similar to that of tumor-prone p53-/- mice. However, while p53-/- mice developed a variety of tumor types, nearly all Gadd45a-/-p53-/- mice developed lymphoblastic lymphoma (LBL), often accompanied by mediastinal masses as is common in human patients with this tumor type. Deletion of Gadd45a in leukemia/lymphoma-prone AKR mice decreased the latency for LBL. These results indicate that Gadd45a may act as modifier locus for T-cell LBL, whereby deletion of Gadd45a enhances development of this tumor type in susceptible mice. Gadd45a is localized to 1p31.1, and 1p abnormalities have been described in T-cell lymphomas. Related human tumor samples did not show Gadd45a deletion or mutation, although changes in expression could not be ruled out.     

Growth of human lymphoma cells in SCID mice.

Two EBV-negative human lymphoma cell lines raised in this laboratory and peripheral blood cells of a patient with large cell lymphoma in leukemic phase were injected intravenously or intraperitoneally into C.B.17 SCID mice. One line (OCI-LY18) was derived from the pleural fluid of a patient with a large cell, immunoblastic malignant lymphoma. Cells of this line are of B cell origin and characterized by multiple rearrangements of the JH locus. The second line (OCI-LY17) was grown from the peripheral blood of a patient with a large cell lymphoma of T-cell phenotype which has characteristic rearrangement of the T-cell receptor beta chain. The cells directly obtained from a patient with large cell non-cleaved malignant lymphoma were of B-cell origin. Animals carrying OCI-LY18 developed large tumor masses within 6-8 weeks of inoculation. The tumors were detected in the intestine, mesentery, retroperitoneum, lymph nodes, spleen, lung and kidney. The masses resembled the primary tumor with respect to histological appearance and immunological phenotype. It was possible to generate secondary cell lines from the tumors found in the inoculated SCID mice. Injection of one of these secondary cell lines into SCID mice resulted in the rapid development of lymphoma and as few as 10 cells were sufficient to establish the disease in the inoculated animals. In contrast cells of OCI-LY17 produced small tumor aggregates that did not appear to progress over time and did not cause death of the animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Positive correlation between in vitro NK activity and in vivo resistance towards AKR lymphoma cells

The possible in vivo relevance of murine natural killer (NK) cells in resistance towards grafted lymphoma was analyzed. The system comprised two AKR T lymphomas having similar behavior as to growth patterns but significant differences in vitro with regard to susceptibility to NK-cell-mediated lysis. These tumor cells were grafted into (AKRxCBA)F₁ hybrids which had been lethally irradiated and then protected with bone marrow of CBA or AKR origin. Some adult animals were thymectomized before irradiation. The F₁ chimeras could be shown to be repopulated with donor cells at the level of peripheral white blood cells and displayed high or low NK levels if coming from CBA and AKR donors respectively. Thymectomy resulted in a significant increase in the final levels of NK activity in the recipients. The in vivo growth patterns of the two AKR lymphomas were in a strikingly positive manner correlated with the NK activities of the recipients. Thus, high NK activity in the recipients resulted in a significant increase in resistance towards the outgrowth of the NK-sensitive AKR lymphoma but had only marginal impact on the relatively NK resistant lymphoma. This was true irrespective of whether or not the recipients were thymectomized before marrow reconstitution and in general the T-deficient mice were thus more resistant towards tumor grafting than their non-thymectomized counterparts. In all, the results would thus further suggest an important role for NK cells in mediating in vivo resistance towards NK-susceptible tumors, and they fail to provide any evidence for T lymphocytes in tumor protection in the same system.     

Potent anti-tumor effects of an anti-CD24 ricin A-chain immunotoxin in vitro and in a disseminated human Burkitt's lymphoma model in SCID mice

A new anti-CD24 immunotoxin (IT), SWA11.dgA, was constructed by coupling the MAb SWA11 via the bivalent linker SMPT to deglycosylated ricin A-chain (dgA). The effects of SWA11.dgA were evaluated in vitro against the B-precursor leukemia cell line REH, the non-B-non-T acute lymphoblastic leukemia cell line NALM-6 and the Burkitt's lymphoma cell lines BL-2 and BL-38. Binding of SWA11 to the CD24 antigen was assessed by flow cytometry demonstrating high affinity of the MAb for all cell lines tested. SWA11.dgA inhibited the protein synthesis of BL-38, NALM-6, REH and BL-2 cells by 50% at concentrations (IC₅₀) of 4.0 × 10⁻¹¹ M, 6.0 × 10⁻¹¹ M, 8.0 × 10⁻¹¹ M and 3.0 × 10⁻⁹ M, respectively. SWA11.dgA was subsequently used for the treatment of disseminated human BL-38 Burkitt's lymphoma in a newly developed SCID mouse model. The mean survival time (MST) of BL-38-bearing SCID mice was extended from 23 days in untreated controls to more than 230 days when 6 μg SWA11.dgA was applied intraperitoneally one day after tumor challenge. All of the animals achieved continuous complete remissions. SCID mice treated with SWA11.dgA 4 days after tumor cell challenge or a reduced dose of SWAII.dgA (67%) also had a significantly extended MST (45.0 and 51.4 days, respectively, as compared to 22.7 and 23.1 days in the controls). We conclude that SWA11.dgA might be of potential use for the treatment of lymphoma in man. © 1996 Wiley-Liss, Inc.     

The ALK inhibitor ASP3026 eradicates NPM-ALK+ T-cell anaplastic large-cell lymphoma in vitro and in a systemic xenograft lymphoma model

NPM-ALK⁺ T-cell anaplastic large-cell lymphoma (ALCL) is an aggressive type of cancer. Standard treatment of NPM-ALK⁺ ALCL is CHOP polychemotherapy. Although patients initially respond favorably to CHOP, resistance, relapse, and death frequently occur. Recently, selective targeting of ALK has emerged as an alternative therapeutic strategy. ASP3026 is a second-generation ALK inhibitor that can overcome crizotinib resistance in non-small cell lung cancer, and is currently being evaluated in clinical trials of patients with ALK⁺ solid tumors. However, NPM-ALK⁺ ALCL patients are not included in these trials. We studied the effects of ASP3026 on NPM-ALK⁺ ALCL cell lines in vitro and on systemic lymphoma growth in vivo. ASP3026 decreased the viability, proliferation, and colony formation, as well as induced apoptotic cell death of NPM-ALK⁺ ALCL cells. In addition, ASP3026 significantly reduced the proliferation of 293T cells transfected with NPM-ALK mutants that are resistant to crizotinib and downregulated tyrosine phosphorylation of these mutants. Moreover, ASP3026 abrogated systemic NPM-ALK⁺ ALCL growth in mice. Importantly, the survival of ASP3026-treated mice was superior to that of control and CHOP-treated mice. Our data suggest that ASP3026 is an effective treatment for NPM-ALK⁺ ALCL, and support the enrollment of patients with this lymphoma in the ongoing clinical trials.     

Immunological studies on mouse mammary tumors. I. Induction of resistance to tumor isograft in C3H/He mice

An isografted tumor MM2, originating from a spontaneous mammary tumor in a C3H/He/mouse, killed 4- to 5-week-old mice within 30 days through intraperitoneal injection of 2 × 10⁴ cells per mouse. It was shown that resistance to the isograft was induced by injection of 1.5 × 10⁵ or 2.0 × 10⁵ tumor cells sensitized with serum (5 μg antibody N) of rabbits immunized with the tumor. Four weeks after injection of the sensitized MM2 cells, 173 C3H/He mice were challenged intraperitoneally with fresh unsensitized MM2 in doses of 5 × 10⁵ to 1 × 10⁶ cells per mouse. Of these, 119 mice survived without any sign of tumor growth while all untreated mice died. The mean survival time for untreated mice was 18.1 days (± 1.7) when inoculated with 5 × 10⁵ cells and 16.2 days (± 1.8) when inoculated with 1 × 10⁶ cells. After the second challenge with the same number of fresh unsensitized cells, 117 out of 119 mice survived without any sign of tumor growth. All of 15 mice randomly selected from these resistant mice were tested for acceptance of skin grafts from normal C3H/He mice. The grafts showed no rejection even 150 days after the second graft. After hyperimmunization of these resistant mice, the serum of the spleen extract taken from the mice inhibited growth of tumors when the tumors were premixed in vitro and injected intraperitoneally into C3H/He mice.     

EFFECTS OF IN VIVO GENE TRANSDUCTION OF ANTI－MDR1 RIBOZYME IN COMBINATION WITH CHEMOTHERAPY ON MULTIDRUG－RESISTANT HUMAN LYMPHOMA GROWTH IN MICE

Chemotherapy is one of the major therapy for the treatment of cancer. However, its effect is often severely affected by the occurrence of multidrug resistant (MDR) of tumor cells. It is imperative to restore MDR for improving therapeutic effect of chemotherapy. It has been proved that over-expression of P-gp is a common mechanism responsible for MDR[1-3]. One practical strategy is to interrupt MDR1 mRNA coding for P-gp by introduction of anti-MDR1 ribozyme into tumor cells. Our previo…     

Endotoxin-stimulated immune response to modified lymphoma cells.

Bacterial endotoxin was administered with iodoacetamide-modified P1798 lymphoma cells to immunize syngenic BALB/cJ mice against this lymphoma to which they are naturally unresponsive. Three or four vaccinations with endotoxin (6.6 mug/injection) alone or the modified cells alone did not produce host resistance. A significant number (30 percent) of mice receiving both endotoxin and modified cells rejected a subsequent implant of viable tumor cells. Even those mice having progressive tumor growth exhibited prolonged survival. High doses of endotoxin given with the modified P1798 cells caused 70-75 percent of the mice to reject the tumor implants. When resistance developed, antibodies reacting with tumor cell membrane were demonstrable. These results indicate that B-lymphocyte stimulators can produce an effective immune response against lymphoma cells.     

Induction of a cytolytic T-cell response in mice with a recombinant adenovirus coding for tumor antigen P815A

We investigated the efficacy of a recombinant adenovirus in inducing a cytolytic T-lymphocyte (CTL) response in mice against tumor antigen P815A, which is present on mouse mastocytoma P815. The recombinant adenoviral vector (Adeno.PIA) contained the sequence coding for the antigenic nonapeptide which binds to the H-2.L^d molecule to form antigen P815A. We verified that murine cells infected in vitro with Adeno.PIA were lysed by an anti-P815A CTL clone. Mice then received a single intradermal injection of Adeno.PIA, and after a few weeks their spleen cells were stimulated in vitro with tumor cells expressing antigen P815A. An anti-P815A CTL response was observed with the spleen lymphocytes of nearly all the mice, providing the lymphocytes were re-stimulated in vitro with cells expressing both P815A and co-stimulatory molecule B7.1. When the stimulatory cells did not express B7.1, a specific CTL response was observed in only 45% of the mice, and it was less intense. The Adeno.PIA viral vector was unable to raise an anti-P815A response in mice that had been previously infected with a recombinant adenovirus carrying the β-galactosidase gene or with a defective adenovirus. We conclude that adenoviral vectors may be very useful for the priming of cytolytic T-cell responses directed against human tumor antigens. Other modes of immunization may be necessary to boost the responses induced with adenoviral vectors. © 1996 Wiley-Liss, Inc.     

Hyaluronan-independent lodgment of CD44+ lymphoma cells in lymphoid organs

We previously found that monoclonal antibodies (MAbs) directed against the constant region of the CD44 molecule block lymph node infiltration of a mouse LB T-cell lymphoma, suggesting a role for this glycoprotein in the LB cell dissemination process. In the present study, we investigated whether LB cells in the local tumor must undergo a change in the CD44 phenotype to be able to migrate to and invade the remote lymph nodes, and if hyaluronic acid (HA), the principal ligand of activated CD44, functions as a mediator in this process. We compared the in vivo behavior of a non-HA-binder LB cell line with that of a constitutive HA-binder HA9 subline. Our results show that the lymphoid organ-infiltrating LB cells express similar levels of pan-CD44 and V4- and V6-containing CD44 variants, as the corresponding cells in the local growth and the cultured LB cells. The tested CD44 phenotype of HA9 cells also remained unchanged during the metastatic process. Even after lymph node infiltration, LB cells remained incapable of binding HA, whereas the HA9 cells retained an efficient HA-binding capacity. The constitutive HA-binder HA9 cells that expressed an approximately 10-fold higher level of pan-CD44 than did the parental LB cells, as well as an elevated level of the V4 and V6 exon products formed a local tumor and invaded both lymph nodes and spleen, as did the parental LB cells, albeit at a much slower rate. Our finding indicates that there is no direct correlation between the amount of CD44 expressed on the cell surface, the HA-binding capacity and tumorigenicity. Moreover, interaction with HA is not obligatory for LB cell localization in the lymphoid organs, and a tight cell-HA interaction, as observed in HA9 cells, does not prevent tumor cell dissemination, although it may retard tumor spread. Int. J. Cancer 71:462-469, 1997. © 1997 Wiley-Liss Inc.     

Ay allele promotes azoxymethane‐induced colorectal carcinogenesis by macrophage migration in hyperlipidemic/diabetic KK mice

The incidence of colorectal cancer has been increasing and is associated with obesity and diabetes. We have found that type 2 diabetes model KK‐A^y/TaJcl (KK‐A^y) mice develop tumors within a short period after treatment with azoxymethane (AOM). However, factors that contribute to the promotion of carcinogenesis have not been clarified. Therefore, we looked at the genetic background of KK‐A^y, including two genetic characteristics of KK/TaJcl (KK) mice and C57BL/6J‐Ham‐A^y/+ (A^y) mice, compared with other non‐obese and non‐diabetic mouse strains C57BL/6J and ICR, and induced colorectal premalignant lesions, aberrant crypt foci (ACF), and tumors using AOM (150 μg/mouse/week for 4 weeks and 200 μg/mouse/week for 6 weeks, respectively). The mice with a diabetes feature, KK‐A^y and KK, developed significantly more ACF, 67 and 61 per mouse, respectively, whereas ICR, A^y, and C57BL/6J mice developed 42, 24, and 18 ACF/mouse, respectively, at 17 weeks of age. Serum insulin and triglyceride levels in KK‐A^y and KK mice were quite high compared with other non‐diabetic mouse strains. Interestingly, KK‐A^y mice developed more colorectal tumors (2.7 ± 2.3 tumor/mouse) than KK mice (1.2 ± 1.1 tumor/mouse) at 25 weeks of age, in spite of similar diabetic conditions. The colon cancers that developed in both KK‐A^y and KK mice showed similar activation of β‐catenin signaling. However, mRNA levels of inflammatory factors related to the activation of macrophages were significantly higher in colorectal cancer of KK‐A^y mice than in KK. These data indicate that factors such as insulin resistance and dyslipidemia observed in obese and diabetic patients could be involved in susceptibility to colorectal carcinogenesis. In addition, increase of tumor‐associated macrophages may play important roles in the stages of promotion of colorectal cancer.     

Ketone supplementation decreases tumor cell viability and prolongs survival of mice with metastatic cancer

Cancer cells express an abnormal metabolism characterized by increased glucose consumption owing to genetic mutations and mitochondrial dysfunction. Previous studies indicate that unlike healthy tissues, cancer cells are unable to effectively use ketone bodies for energy. Furthermore, ketones inhibit the proliferation and viability of cultured tumor cells. As the Warburg effect is especially prominent in metastatic cells, we hypothesized that dietary ketone supplementation would inhibit metastatic cancer progression in vivo. Proliferation and viability were measured in the highly metastatic VM‐M3 cells cultured in the presence and absence of β‐hydroxybutyrate (βHB). Adult male inbred VM mice were implanted subcutaneously with firefly luciferase‐tagged syngeneic VM‐M3 cells. Mice were fed a standard diet supplemented with either 1,3‐butanediol (BD) or a ketone ester (KE), which are metabolized to the ketone bodies βHB and acetoacetate. Tumor growth was monitored by in vivo bioluminescent imaging. Survival time, tumor growth rate, blood glucose, blood βHB and body weight were measured throughout the survival study. Ketone supplementation decreased proliferation and viability of the VM‐M3 cells grown in vitro, even in the presence of high glucose. Dietary ketone supplementation with BD and KE prolonged survival in VM‐M3 mice with systemic metastatic cancer by 51 and 69%, respectively (p < 0.05). Ketone administration elicited anticancer effects in vitro and in vivo independent of glucose levels or calorie restriction. The use of supplemental ketone precursors as a cancer treatment should be further investigated in animal models to determine potential for future clinical use.     

Liver endothelial cells participate in T-cell-dependent host resistance to lymphoma metastasis by production of nitric oxide in vivo

Tumor growth and metastasis of IacZ-transduced murine lymphoma ESbL cells inoculated into syngeneic DBA/2 mice are characterized by a transient plateau phase with a constant tumor diameter and low metastatic load, indicating a host response against the tumor. Here we show that endothelial cells participate in a T-cell-dependent, anti-metastatic response by producing NO in situ. Liver endothelial cells were isolated and examined directly ex vivo without further manipulation. NO production in liver endothelial cells reached the highest level during the plateau phase but declined toward the end of it, followed by an overall breakdown of host response, leading to progressive tumor growth and high load of liver metastasis. Mice subjected to anti-tumor immunization and subsequent challenge with a tumorigenic dose of ESbL-IacZ cells showed, in comparison to non-immunized challenged controls, reduced liver metastasis and increased endothelial NO production. Adoptive transfer of anti-tumor immune spleen cells from semi-allogeneic B10.D2 mice into tumor-bearing animals during the plateau phase caused a regression of primary tumor and metastases, together with a preservation of the high level of NO synthesis in endothelial cells. In immuno-incompetent (SCID) mice, tumor growth and metastasis were progressive and there was no endothelial NO response. Pre-immunization of immuno-competent mice with both live and irradiated tumor cells at different sites of the body led to an induction of NO production by liver endothelial cells. These results reveal a novel role of endothelial cells in the suppression of lymphoma metastasis in the liver. The inducible endothelial cell NO response is apparently dependent and induced by mature T lymphocytes.     

Dietary supplementation with curcumin enhances metastatic growth of Lewis lung carcinoma in mice

Our study investigated the effects of dietary supplementation with curcumin [(1E,6E)‐1,7‐bis(4‐hydroxy‐3‐methoxyphenyl)‐1,6‐heptadiene‐3,5‐dione] on spontaneous metastasis of Lewis lung carcinoma (LLC) in C57BL/6 mice. Mice were fed with the AIN93G control diet or with the diet supplemented with 2 or 4% curcumin for 5 weeks at which time they were injected subcutaneously with 2.5 × 10⁵ viable LLC cells. The subcutaneous primary tumor was surgically removed when it reached ～ 8 mm in diameter, and the experiment was terminated 10 days after the surgery. There was no difference in pulmonary metastatic yield among the groups. Curcumin supplementation at either dietary level did not significantly increase the size of metastatic tumors; however, the combined data from both curcumin groups showed that curcumin treatment increased metastatic tumor cross‐sectional area by 46% (p < 0.05) and volume by 70% (p < 0.05) compared to the controls. Curcumin supplementation increased plasma concentrations of angiogenic factors angiogenin (p < 0.05), basic fibroblast growth factor (p < 0.05) and vascular endothelial growth factor (p < 0.05), as well as inflammatory cytokines interleukin‐1β (p < 0.05) and monocyte chemotactic protein‐1 (p < 0.05), compared to the controls. These results demonstrate that curcumin does not prevent metastasis and indicate that it can enhance metastatic growth of LLC in mice, perhaps through upregulation of angiogenesis and inflammation.     

Growth and treatment of Ehrlich tumor in mice with alloxan-induced diabetes.

Hypoglycemia and hypoinsulinemia accompanied i.p. or i.m. growth of the Ehrlich tumor in CBA/H and BALB/c mice. Simultaneously, insulin accumulated in the ascitic fluid of tumor-bearing mice. In hosts rendered diabetic by means of alloxan, the tumor decreased the blood glucose almost to the level seen in nondiabetic mice. Tumor growth was retarded in diabetic hosts, but cells from such tumors, transplanted into secondary diabetic recipients, grew faster than in their primary diabetic hosts, similarly to "nondiabetic" tumor cells growing in nondiabetic hosts. This phenomenon of "adaptation" of the tumor to the diabetic state was prevented if diabetic tumor-bearing mice were daily treated with insulin. The tumor did not grow in all diabetic recipients; the frequency of takes correlated with severity of the diabetes, i.e., with the dose of alloxan given to induce it. The greater the dose, the less mice accepted the tumor. Insulin injection into diabetic tumor-bearing mice promoted the tumor growth. Simultaneous treatment of diabetes and the tumor afforded the best antitumor effect.     

Tolerance to the anti-metastatic effect of lipopolysaccharide against liver metastasis in mice

We describe the involvement of endotoxin tolerance in the refractoriness of its anti-metastatic effect against murine syngeneic tumors. Three i.v. administrations of LPS at intervals of 4 days after tumor inoculation inhibited liver metastasis of L5178Y-ML25 cells, whereas 3 consecutive i.v. administrations of LPS showed only a slight suppressive effect. Multiple i.v. administrations of LPS, synthetic lipid A, its synthetic derivative DT-5461, Staphylococcus aureus (S. aureus) BioParticles or Staphylococcal enterotoxin B (SEB) on days 1, 5 and 9 after tumor inoculation inhibited liver metastasis of T-lymphoma cells in normal mice. The anti-metastatic effects of LPS, synthetic lipid A or DT-5461 but not S. aureus BioParticles or SEB were diminished in mice injected with LPS at daily intervals for 7 days before tumor inoculation. Mice receiving 3 consecutive i.v. administrations of LPS at daily intervals exhibited suppression of LPS-induced production of endogenous tumor necrosis factor-α (TNF-α), tumoricidal activity of macrophages, and natural-killer (NK) activity of splenocytes when compared with those of normal mice. Macrophages from mice receiving consecutive daily i.v. administrations of LPS for 3 days showed reduction of LPS-induced tyrosine phosphorylation of several intracellular proteins, including p42^mapk/ERK2 when compared with that of the cells obtained from normal mice. These data suggest that the LPS-induced anergic state of monocytes/macrophages plays a crucial role in endotoxin tolerance with respect to the metastasis of T lymphoma in the liver. © 1996 Wiley-Liss, Inc.     

Histone deacetylase inhibitors in lymphoma and solid malignancies

Histone deacetylase inhibitors (HDACi) are a new class of antineoplastic agents with demonstrable preclinical antitumor activity in both in vitro and in vivo studies in a wide range of malignancies. Based on these preclinical findings, in recent years HDACi have undergone a rapid phase of clinical development with many HDACi entering Phase I–III clinical trials, both as single agents and in combination with other therapies. Favorable clinical responses have been demonstrated in cutaneous T-cell lymphoma with emerging evidence of clinical activity in other types of lymphoma and, to date, a good toxicity profile. Solid tumor responses to single agent and combination therapies have also been reported, paving the way for larger studies in this field. In this review we discuss the recent advances in the clinical development of HDACi and their current therapeutic role in lymphoma and solid malignancies.