Interaction of IFN α/β with host cells essential to the early inhibition of friend erythroleukemia visceral metastases in mice

We have previously shown that an intact immune system was essential to the increase in survival time of IFN-α/β-treated mice injected i.v. with an IFN-α/β-resistant line of Friend erythroleukemia cells (FLC) highly metastatic to the liver and spleen. Here, we have investigated the early interactions of IFN α/β with host cells prior to the development of the immune response. IFN α/β treatment resulted in 50- to 100-fold inhibition of FLC multiplication in the liver and spleen of normal DBA/2 mice shortly after tumor inoculation, as evaluated by colony formation in agarose. IFN treatment was far less effective in inhibiting the multiplication of FLC in the livers of NK-cell-deficient DBA/2 beige mice, or in immunocompetent DBA/2 mice treated with antibody to asialo GMI, or silica, or in mice subjected to sub-lethal irradiation. Injection of antibody to CD4 or CD5 did not affect the early inhibitory action of IFN α/β on FLC multiplication but did decrease survival time. Light- and electron-microscope examination of the livers of IFN-treated, FLC-injected mice confirmed the early inhibition of FLC multiplication in the liver and spleen. Our results indicate that IFN α/β inhibits the development of FLC visceral metastases by acting first on host cells, such as NK cells and macrophages, and then continues to act in consort with the developing immune response. © 1994 Wiley-Liss, Inc.     

The essential role of endogenous IFN α/β in the anti-metastatic action of sensitized T lymphocytes in mice injected with friend erythroleukemia cells

Adoptive transfer of splenic T lymphocytes from DBA/2 mice immunized against Friend erythroleukemia cells (FLC) inhibited the development of visceral metastases and increased the survival time of DBA/2 mice challenged i.v. with parental FLC 24 hr to 2 months later. Immune spleen cells were ineffective in mice pre-treated with potent neutralizing antibody to mouse IFN α/β (but not to IFN γ), demonstrating the essential participation of endogenous IFN α/β in the inhibitory action of immune T lymphocytes against FLC metastases. These findings suggest that the reported inability of immune T lymphocytes to exert an anti-FLC effect in immunodeficient DBA/2 mutant beige (bg/bg) mice (unless these mice had also been treated with IFN α/β), may have been due to lower levels of endogenous IFN α/β in DBA/2 bg/bg mice than in normal DBA/2 +/bg mice. Experimental results in support of this hypothesis are presented. © 1995 Wiley-Liss, Inc.     

Synergistic anti-tumor effects of combined IL-1/IFN-alpha/beta therapy in mice injected with metastatic Friend erythroleukemia cells

Peritumoral injection of relatively low doses of either mouse interferon (IFN)-alpha/beta (10,000-20,000 units/injection) or of recombinant human interleukin-1 (IL-1) beta (125-250 ng/injection) in mice transplanted s.c. with Friend erythroleukemia cells (FLC) resulted in some inhibition of primary tumor growth, inhibition of liver and splenic metastases and increased survival time. A synergistic anti-tumor effect was observed in mice injected with both IL-1 and IFN-alpha/beta. Highly purified mouse IFN-beta also exerted a synergistic anti-tumor effect when combined with IL-1-beta in mice injected with FLC. The anti-tumor action of IL-1/IFN was markedly reduced in mice treated with antibodies to CD4 antigens. Antibodies to asialo-GM1 also diminished the anti-tumor effect by the combined cytokine treatment. The combined IL-1/IFN therapy was effective in NK-deficient bg/bg mice, although the extent of the anti-tumor response in these mice was less than that observed in bg/+mice.     

Importance of interferon alpha in the resistance of allogeneic C57B1/6 mice to the multiplication of Friend erythroleukemia cells in the liver

Friend erythroleukemia cells (FLC) (H-2d) injected intravenously multiply extensively in the livers of syngeneic DBA/2 mice and not at all in the livers of allogeneic C57B1/6 mice. Our results indicate that interferon alpha (IFN-alpha) is an important factor in the resistance of allogeneic mice to the multiplication of FLC in the liver. (a) After i.v. inoculation of FLC there was an inverse correlation between the presence of IFN-alpha in the serum and the capacity of FLC to multiply in the liver. Thus, all 44 FLC-injected adult C57B1/6 mice had circulating IFN-alpha and FLC did not multiply in the liver of any of the mice. Interferon was not detected in the serum of 83% of 41 FLC-injected DBA/2 mice (and was found only at a low titer in 17% of the mice) and FLC multiplied in the liver of all mice. (b) FLC did multiply in the livers of newborn C57B1/6 mice and in the livers of irradiated adult C57B1/6 mice, and IFN-alpha was not detected in their sera. In contrast, after i.v. inoculation of FLC, IFN-alpha was detected in the sera of 3-week-old and athymic nu/nu adult C57B1/6 mice while FLC failed to multiply in the liver. (c) FLC also induced IFN-alpha in congenic B10.D2 (H-2d) mice and FLC did not multiply in the liver. We suggest that, depending on the site of tumor implantation, different host mechanisms have various degrees of importance in controlling the growth and/or rejection of allogeneic tumor cells, and that IFN-alpha is particularly important when FLC are injected i.v.     

Interferon treatment markedly inhibits the development of tumor metastases in the liver and spleen and increases survival time of mice after intravenous inoculation of Friend erythroleukemia cells

To investigate the effect of interferon treatment on the development of tumor metastases, DBA/2 mice were injected i.v. with 2 X 10(6) Friend erythroleukemia cells (FLC) (equivalent to about 5 X 10(5) LD50). FLC multiplied rapidly in the liver and spleen and all untreated or control treated mice died between 7 and 12 days. Daily treatment of mice with potent preparations of mouse interferon alpha/beta was initiated 3 to 72 hr after i.v. inoculation of tumor cells, at times when FLC were already present in the liver and spleen. Interferon treatment resulted in a 100 to 1,000-fold inhibition of the multiplication of FLC in the liver and spleen and a marked increase in mean survival time. Small numbers of tumor cells persisted in the liver and spleen in some interferon-treated mice and could be recovered by bioassay several weeks after tumor inoculation. Most interferon-treated mice died with tumor in the ensuing months. Three of 34 interferon-treated mice were considered cured as they were alive at 386, 325 and 284 days after tumor inoculation. Daily treatment of tumor-inoculated mice with human recombinant interferons alpha D and alpha BDDD, which had antiviral activity on mouse cells in culture, also increased the survival time of mice injected i.v. with FLC. The use of the interferon-resistant 3C18 line of FLC suggests that the marked inhibition of development of established liver and spleen metastases was not due to a direct effect of interferon on the tumor cells, but was host-mediated.     

Inhibition of mouse alpha/beta-interferon of the multiplication of alpha/beta-interferon-resistant Friend erythroleukemia cells cocultured with mouse hepatocytes

Administration of alpha/beta-interferon (IFN) exerts a marked antitumor effect in DBA/2 mice given injections i.v. of large numbers of IFN-alpha/beta-resistant erythroleukemia cells (FLC). To investigate the possible mechanisms of FLC tumor inhibition in the liver of interferon-treated mice, we developed an in vitro model consisting of a coculture of IFN-alpha/beta-resistant 3Cl8 FLC and syngeneic mouse hepatocytes. Whereas IFN-alpha/beta did not inhibit the multiplication of 3Cl8 FLC cultivated alone, it effectively inhibited the multiplication of 3Cl8 FLC in coculture with hepatocytes. The inhibitory effect was directly proportional to the amount of IFN-alpha/beta added to the cocultures, and more than 90% inhibition of FLC multiplication was noted with 1.6 x 10(5) IU/ml of IFN-alpha/beta on Day 3 of coculture. When FLC were separated from the monolayer of hepatocytes by a pored membrane (0.4 microns), the inhibitory effect on FLC proliferation was unchanged, indicating that a soluble factor(s) released from IFN-treated hepatocytes was most important in the inhibition of FLC multiplication. An inhibitory activity of FLC multiplication was detected only in the conditioned medium of IFN-treated hepatocytes but not in the conditioned medium of control hepatocytes nor in extracts of IFN-treated or control hepatocytes. The inhibitory factor(s) in the conditioned medium of IFN-treated hepatocytes was retained by an ultrafiltration membrane (Mr cut off 10,000), and its activity was completely abrogated by trypsin digestion. Its stability to treatment with 1 M acetic acid as well as lack of correlation between the antiproliferative effect and the amount of L-arginine in the medium distinguished this factor(s) from liver arginase which was also found to be a potent inhibitor of FLC multiplication in vitro. The inhibitory factor(s) was also distinguishable in its biological activity from IFN gamma, interleukin 1 alpha and beta, and transforming growth factor beta 1 and beta 2. These results suggest the possibility that the inhibitory effect of IFN-alpha/beta on the development of 3Cl8 FLC in the livers of IFN-treated mice may be mediated by an IFN-induced inhibitor of FLC multiplication.     

Anti-tumor effects of interferon in mice injected with interferon-sensitive and interferon-resistant Friend leukemia cells. VI. Adjuvant therapy after surgery in the inhibition of liver and spleen metastases

Adult DBA/2 mice were injected s.c. with the highly malignant, interferon-resistant 3C18 line of Friend erythroleukemia cells (FLC). Eight or 9 days after established s.c. tumors had developed, the primary tumor was excised and mice were treated i.p. with either mouse interferon alpha/beta or a control preparation. At the time of surgery, mice already had tumor cells in the liver. All control-treated mice died in the ensuing 2 weeks with extensive tumor metastases in the liver and spleen. Interferon treatment resulted in an inhibition of the development of liver and spleen metastases and a markedly increased survival time. We conclude that interferon alpha/beta is effective as adjuvant therapy after surgery for metastatic disease in mice.     

Effectiveness of mouse interferon alpha/beta compared to single-agent chemotherapy in increasing survival time of mice after intravenous inoculation of Friend erythroleukemia cells

DBA/2 mice received an iv injection of 2 X 10(6) Friend erythroleukemia cells (FLCs; approximately equal to 4 X 10(5) lethal dose50), which multiplied rapidly in the liver and spleen and killed all untreated or control treated mice between 7 and 12 days. Daily interferon (IFN) treatment resulted in a very marked increase in survival time and apparent cure of 4 of 22 tumor-inoculated mice. In contrast, treatment of tumor-injected (iv) mice with cyclophosphamide, 5-fluorouracil, and methotrexate increased survival time by only a few days; and treatment of mice with cisplatin, vincristine, doxorubicin, bleomycin, or etoposide was ineffective. However, when FLCs were injected ip, both cytostatic drugs and IFN exerted an antitumor effect. We conclude that IFN alpha/beta was particularly effective in inhibiting the development of liver and spleen metastases and in increasing mouse survival time after iv inoculation of FLCs.     

Biologic and biochemical differences between in vitro and in vivo passaged Friend erythroleukemia cells. I. Tumorigenicity and capacity to metastasize

Cloned interferon-sensitive (745) and interferon-resistant (3Cl-8) Friend erythroleukemia cells (FLC) passaged in vitro, are not very tumorigenic when first injected intraperitoneally (i.p.) into syngeneic DBA/2 mice although they do form solid tumors when injected subcutaneously (s.c.). By serially passaging FLC (either 745 or 3Cl-8 cells) i.p. in DBA/2 mice, we obtained two different FLC lines capable of growing i.p. and inducing tumor ascites. The s.c. injection of DBA/2 mice with these in vivo passaged FLC resulted in tumor metastases in the liver and spleen, whereas metastases were not observed in mice inoculated s.c. with in vitro passaged FLC. The capacity of in vivo passaged FLC to metastasize was acquired after several i.p. passages. This highly malignant behavior was a stable characteristic of these cells. All the clones derived from in vivo passaged FLC and passaged more than 14 times in vitro induced hemorrhagic ascites when injected i.p., and metastasized to the liver and spleen when injected s.c. The phenotype of sensitivity or resistance to the inhibitory effect of alpha/beta mouse interferon on virus replication and cell multiplication was conserved during serial i.p. passages and maintained in the clones derived from in vivo passaged cells. These FLC showed a decreased capacity to differentiate in vitro upon treatment with dimethylsulfoxide (DMSO) and a reduced production of Friend leukemia virus with respect to the original clones passaged in vitro.     

Anti-tumor effects of interleukin-2 and interleukin-1 in mice transplanted with different syngeneic tumors

We have studied the anti-tumor effects of human recombinant IL-2, alone or in association with LAK cells, in mice transplanted subcutaneously (s.c.) with the following syngeneic tumors: highly metastatic Friend leukemia cells (FLC), nonmetastatic FLC, lymphoma RBL-5 cells and HeJ16 fibrosarcoma cells. In these tumor models, peri-tumoral injections of IL-2 were more effective in inhibiting tumor growth than a systemic treatment. Although s.c. IL-2 treatment resulted in marked inhibition of tumor growth in mice injected s.c. with highly metastatic FLC, it was not effective in inhibiting growth of FLC in the liver and spleen. IL-2 therapy was more effective at increasing survival time in mice transplanted with non-metastatic FLC or with RBL-5 cells. In mice transplanted with HeJ16 fibrosarcomas, s.c. IL-2 treatment resulted in highly significant anti-tumor effect and survival of 70% of tumor-injected mice. No general correlation was found between in vitro sensitivity or resistance to the cytolytic activity of LAK cells and the anti-tumor effects observed in vivo. Subcutaneous injection of IL-1 beta in mice transplanted with highly metastatic FLC resulted in a marked increase in survival time and inhibition of metastatic tumor growth in liver and spleen. Combined treatment of IL-1 beta and IL-2 produced a synergistic anti-tumor effect: 60% of mice injected with highly metastatic FLC survived. Combined IL-1/IL-2 treatments exerted no anti-tumor activity either in DBA/2 mice injected with antibody to Thy 1.2 antigen or in nude mice, indicating that T cells play important roles during IL-1/IL-2 therapy. In vitro treatment of FLC with IL-1 beta resulted in a slight inhibition of cell multiplication, whereas even high doses of IL-2 did not affect FLC multiplication. Our results indicate that local combined treatments with IL-1 and IL-2 can induce potent, host-dependent (T cell-mediated) anti-tumor effects against highly malignant tumors.     

αv-Integrins in human melanoma: Gain of αvβ3 and loss OF αvβ5 are related to tumor progression in situ but not to metastatic capacity of cell lines in nude mice

We investigated the expression of α_v-integrins in different stages of human cutaneous melanocytic tumor progression. We observed that α_vβ₅ was the α_v-integrin expressed in all common nevocellular nevi, in 78% of dysplastic nevi, in 63% of early primary melanomas, in 43% of advanced primary melanomas, and in 33% of melanoma metastases. Hence, loss of α_vβ₅ expression was related to melanocytic tumor progression. In line with earlier reports, α_vβ₃ was exclusively detected in advanced primary melanomas and metastases (24% and 50% respectively). Staining with anti-α_v monoclonal antibodies (MAbs) in lesions where both α_vβ₃ and α_vβ₅ were absent showed that alternative α_v-integrins were expressed in advanced primary melanomas and metastases. By FACS analysis, we determined expression of α_vβ₅ and α_vβ₃ in 4 human melanoma cell lines with different metastatic capacities after s.c. inoculation into nude mice. One of the non-metastatic and both highly metastatic cell lines expressed α_vβ₅ at their surface. Surprisingly, α_vβ₃ was detected exclusively in the non-metastatic cell lines. Absence of α_vβ₃ in the highly metastatic cell lines was confirmed by lack of immunoprecipitation from ³⁵S-methionine-labeled cells and by absence of immunohistochemical staining on primary and metastatic xenograft lesions. Our findings indicate that α_vβ₅ expression is often lost in advanced stages of melanocytic tumor progression in situ, while α_vβ₃ is acquired, but that a decrease in α_vβ₅ and an increase in α_vβ₃ expression are not necessarily related to the metastatic behavior of human melanoma cells in nude mice. © 1995 Wiley-Liss, Inc.     

Murine natural antitumor antibodies. III. Interferon treatment of a natural killer-resistant lymphoma: augmentation of natural antibody reactivity and susceptibility to in vivo natural resistance

Treatment of a natural killer cell-resistant (NKR) DBA/2 lymphoma with L-cell interferon (IFN) enhanced its reactivity to serum natural antibody in vitro in cytolysis and absorption studies and increased the in vivo acquisition of natural and antitumor antibody in the peritoneal cavity. The IFN effects were both time- and dose-dependent. In vitro IFN-treated, [131I]5-iodo-2'-deoxyuridine-labeled tumor cells, when injected ip into normal syngeneic mice, were more rapidly eliminated than were untreated control cells. IFN treatment of the NKR tumor decreased "cold-target" inhibition of NK lysis and did not alter binding or lysis by macrophages. These findings indicated that the enhancement of natural resistance to the IFN-treated tumor did not involve NK cells or macrophages and suggested that IFN may enhance host antitumor resistance by increasing tumor reactivity to antibody.     

Potent systemic therapy of multiple myeloma utilizing oncolytic vesicular stomatitis virus coding for interferon-β

Multiple myeloma (MM) is an incurable malignancy of plasma secreting B cells disseminated in the bone marrow. Successful utilization of oncolytic virotherapy for myeloma treatment requires a systemically administered virus that selectively destroys disseminated myeloma cells in an immune-competent host. Vesicular stomatitis virus (VSV)-expressing interferon-β (IFNβ) is a promising new oncolytic agent that exploits tumor-associated defects in innate immune signaling pathways to destroy cancer cells specifically. We demonstrate here that a single, intravenous dose of VSV coding for IFNβ (VSV-IFNβ) specifically destroys subcutaneous and disseminated 5TGM1 myeloma in an immune-competent myeloma model. VSV-IFN treatment significantly prolonged survival in mice bearing orthotopic myeloma. Viral murine IFNβ expression further delayed myeloma progression and significantly enhanced survival compared with VSV-expressing human IFNβ. Evaluation of VSV-IFNβ oncolytic activity in human myeloma cell lines and primary patient samples confirmed myeloma-specific oncolytic activity, but revealed variable susceptibility to VSV-IFNβ oncolysis. The results indicate that VSV-IFNβ is a potent, safe oncolytic agent that can be systemically administered to target and destroy disseminated myeloma effectively in immune-competent mice. IFNβ expression improves cancer specificity and enhances VSV therapeutic efficacy against disseminated myeloma. These data show VSV-IFNβ to be a promising vector for further development as a potential therapy for the treatment of MM.     

Inhibition of Friend erythroleukaemia-cell tumours in vivo by a synthetic analogue of prostaglandin E2.

The effect of 16,16-dimethyl-PGE2-methyl ester (di-M-PGE2), a long-acting synthetic analogue of prostaglandin E2, on the replication of Friend erythroleukaemia cells (FLC) in vivo has been studied. Pre-treatment in vitro of both undifferentiated and differentiated FLC with di-M-PGE2 (1 microgram/ml) did not alter rates of tumour appearance or growth, but increased the median survival of DBA/2J mice. Systemic administration of di-M-PGE2 (10 microgram/mouse/day) was not toxic to the mice, but significantly inhibited tumour growth and increased median survival in mice injected s.c. with undifferentiated FLC. These effects of di-M-PGE2 were much more pronounced in mice receiving differentiated (DMSO-treated) FLC. In this latter group, the appearance of tumour was also significantly delayed by di-M-PGE2. The different effects of di-M-PGE2 treatment on tumours derived from undifferentiated and differentiated cells suggest that the analogue is acting directly on tumour-cell replication rather than on factors related to the host response.     

Immune-mediated arrest and reversal of established visceral metastases in athymic mice.

The metastasizing MDAY-D2 tumor of DBA/2 mice disseminates in BALB/c allogeneic athymic nude (nu/nu) mice in a manner identical to that observed in the syngeneic host. Both the kinetics and organ distribution pattern of metastases from s.c. implants of MDAY-D2 are routinely predictable at any given tumor dose. BALB/c heterozygote (nu/+) litter-mates reject MDAY-D2 grafts on the basis of the multiple minor histocompatibility differences that exist between DBA/2 and BALB/c mice. The in vitro cell-mediated cytotoxic response detected in tumor-bearing BALB/c nu/+ mice is "low grade" (isotope release is approximately 40 to 50% by 24-hr 111-indium-8-hydroxyquinoline assay and approximately 6 to 8% by 6-hr 51Cr assay) and yet correlates directly with tumor rejection. BALB/c nu/nu mice can be protected against MDAY-D2 by previous reconstitution with lymphoid cells from normal or MDAY-D2-sensitized BALB/c nu/+ mice. In addition, surgically documented, established visceral metastases in BALB/c nu/nu mice can be arrested and regressed by the adoptive transfer of MDAY-D2-sensitized BALB/c nu/+ spleen cells. This represents one of the few models where established metastases have been immunotherapeutically regressed. As such, the MDAY-D2 BALB/c nu/nu mouse model offers unique advantages for studying the role of the immune system in regulating the metastatic process.     

Adoptive immune therapy in mice bearing poorly immunogenic metastases, using T lymphocytes stimulated in vitro against highly immunogenic mutant sublines

MDW3, a highly immunogenic and non-tumorigenic (tum-) mutant of the poorly immunogenic metastatic murine tumor called MDAY-D2, has been employed in an immune therapy scheme for the treatment of widespread established visceral MDAY-D2 metastases in syngeneic mice. MDW3 was selected from a mutagenized population of MDAY-D2 cells for the ability to grow in the presence of toxic concentrations of wheat-germ agglutinin (WGA) in vitro. The mutant expresses a common tumor-associated antigen (TAA) present on MDAY-D2 as well as a new antigen whose presence enhances the anti-TAA cell-mediated immune response in vivo and in mixed lymphocyte tumor cultures (MLTC) in vitro. For immune therapy, spleen cells from DBA/2 mice which had rejected an inoculum of MDW3 cells were restimulated in MLTC and injected i.v. into MDAY-D2 tumor-bearing mice. Two protocols were used. In the first, mice were given an i.v. injection of 10(3) MDAY-D2 cells ("artificial metastasis") and subsequently treated with 400 R whole-body irradiation and MDW3-stimulated T cells. Such mice had a 75% long-term survival rate, whereas 400 R alone, or no treatment, resulted in 25% and 0% long-term survivors, respectively. In the second protocol, treatment of mice bearing a 12-day-old subcutaneous MDAY-D2 tumor by surgical removal of the solid tumor, 400 R whole-body irradiation, and systemic administration of MDW3-stimulated spleen cells, resulted in a 75-100% survival rate, whereas omitting any part of the treatment resulted in 0-50% survival rates. The treatment increased splenic anti-TAA CTL activity, and the mice acquired immunity against the new antigen on MDW3, suggesting that the injected lymphocytes were proliferating in the host. The optimal combination of resection, whole-body irradiation and passive infusion of MDW3-stimulated spleen cells was ineffective when used on mice bearing a tumor-antigen-loss variant of MDAY-D2, suggesting that success of our immune therapy protocol required specific recognition of the tumor-associated antigen of MDAY-D2.     

Lymphotoxin‐β receptor activation by lymphotoxin‐α1β2 and LIGHT promotes tumor growth in an NFκB‐dependent manner

Lymphotoxin beta receptor (LTβR) activation on mouse fibrosarcoma cells (BFS‐1) results in enhanced solid tumor growth paralleled by increased angiogenesis induced by the expression of pro‐angiogenic CXCL2. In our study, we demonstrate that both functional ligands of the LTβR, namely LTα₁β₂ and LIGHT, are involved in the activation of LTβR in solid fibrosarcomas. To identify whether the lymphocyte population is involved in the activation of LTβR in these fibrosarcoma tumors, we used conditional LTβ‐deficient mice that specifically lack LTβ expression either on T cells (T‐LTβ^?/?) or on B cells (B‐LTβ^?/?). Solid tumor growth was reduced in both mouse strains when compared to tumor growth in wild‐type mice, indicating the participation of both T and B host lymphocytes in the activation of LTβR in these tumors. Tumor growth was also reduced in LIGHT‐deficient mice, suggesting a contribution of this ligand to the activation of LTβR in BFS‐1 fibrosarcomas. LTβR signaling can involve IκBα and/or NFκB‐inducing kinase (NIK) for subsequent NFκB activation in different types of cells. Expression of a dominant negative form of IκBα or of a dominant negative mutant of NIK resulted in decreased activation of NFκB signaling and reduced expression of pro‐angiogenic CXCL2 in vitro. Moreover, expression of dominant negative form of NIK or an IκBα repressor in these fibrosarcoma cells resulted in reduced solid tumor growth in vivo, suggesting that both IκBα and NIK are involved in pro‐angiogenic signaling after LTβR activation. Our data support the idea that the ablation of LTβR signaling should be considered for cancer treatment.     

Cancer therapy with major histocompatibility complex‐deficient and interferon β‐producing myeloid cells derived from allogeneic embryonic stem cells

We previously established a method to generate myeloid cells with a proliferative capability from pluripotent stem cells and designated them iPS‐ML. Human iPS‐ML cells share features with physiological macrophages including the capability to infiltrate into cancer tissues. We observed therapeutic effects of human iPS‐ML cells expressing interferon β (iPS‐ML/interferon (IFN)‐β) in xenograft cancer models. However, assessment of host immune system‐mediated therapeutic and adverse effects of this therapy is impossible by xenograft models. We currently evaluated the therapeutic effects of a mouse equivalent of human iPS‐ML/IFN, a mouse embryonic stem (ES) cell‐derived myeloid cell line producing IFN (ES‐ML/IFN). The ES‐MLs producing IFN‐β (β‐ML) and IFN‐γ (γ‐ML) and originating from E14 ES cells derived from the 129 mouse strain (H‐2^b) were generated, and the MHC (H‐2K^b, D^b, and I‐A^b) genes of the ES‐ML/IFN were disrupted using the clustered regularly interspaced short palindromic repeats (CRISPR)/CAS9 method. We used the ES‐ML/IFN to treat allogeneic BALB/c mice (H‐2^d) transplanted with Colon26 cancer cells. Treatment with β‐ML but not with γ‐ML cells repressed the growth of colon cancer in the peritoneal cavity and liver. The transferred ES‐ML/IFN infiltrated into cancer tissues and enhanced infiltration of T cells into cancer tissues. ES‐ML/IFN therapy increased the number of immune cells in the lymphoid organs. Sensitization of both cancer antigen‐specific CD8⁺ T cells and natural killer (NK) cells were enhanced by the therapy, and CD8⁺ T cells were essential for the therapeutic effect, implying that donor MHC‐deficient β‐ML exhibited a therapeutic effect through the activation of host immune cells derived from allogeneic recipient mice. The results suggested the usefulness of HLA‐deficient human iPS‐ML/IFN‐β cells for therapy of HLA‐mismatched allogeneic cancer patients.     

The capacity of human malignant B-lymphocytes to disseminate in scid mice is correlated with functional expression of the fibronectin receptor α5β1 (CD49E/CD29)

The α₅β integrin (CD49e/CD29), a heterodimeric membrane protein, is the “classical” fibronectin receptor on many cell types. During B-cell ontogeny, expression of the α₅-subunit is developmentally regulated. The αβ₁ is decisive for migration on fibronectin substrate and very likely cooperates with other adhesion molecules in transvascular trafficking. To test whether α₅β influences local growth vs. disseminative spread of neoplastic B-cells in vivo, human B-cell lines mimicking different maturational stages were transferred s.c. into severe combined immunodeficiency (SCID) mice and examined for α₅β expression and for adherence on fibronectin substrate in vitro and ex vivo. All cell lines were locally tumorigenic. Dissemination was observed in all animals carrying Nalm-6 tumors, in one animal with a BL 60 and in 2 mice carrying a Raji tumor. By contrast, Daudi, BJAB and U266 tumors did not disseminate. As evidenced by immunohistochemistry and flow cytometry, all lines and their tumors were to various extents β₁-positive but showed considerable differences in α₅ expression. The functional surface expression of α₅β₁correlated with fibronectin adherence of the lines. Daudi expressed α₅β₁ in a non-functional configuration which was rendered functional only upon applying high concentrations of Mg⁺⁺ and Mn⁺⁺. B-cell lines functionally expressing α₅β₁ at high or moderate levels disseminated in SCID mice while α₅-negative lines and Daudi did not. These results support the conclusion drawn from an earlier in situ analysis of human B-cell lymphomas/ leukemias that the α₅β₁ integrin contributes to the disseminative phenotype of malignant B cells.