Antitumor Effect of Interleukin-1β in the Double Grafted Tumor System

The antimetastatic effect of recombinant human interleukin-1β (rIL-1β) in a new experimental mouse model was studied. Intratumoral administration of IL-1β strongly inhibited the growth of Meth-A solid tumors in male BALB/c mice and led to a complete regression of tumors and resistance to reinoculated tumor. Subsequently, the anti-metastatic effect of IL-1β was examined in the double grafted tumor system, in which mice first received simultaneous intradermal inoculations of Meth-A in both right (10⁶ cells) and left (2×10⁵ cells) flanks and were then injected with 0.2 μg of IL-1β in the right tumor on days 3, 4 and 5. IL-1β significantly inhibited the growth of the left, non-treated tumor. When mice received only an inoculation of Meth-A (2×10⁵ cells) in the left flank and were injected subcutaneously with IL-1β into the right flank on day 3 (single tumor system), there was no inhibition of the growth of the left, non-treated tumor. These findings suggest that intratumoral IL-1β immunotherapy in one region has an effect on tumor growth in another region. Immunized spleen cells were taken from mice which had been cured by the intratumoral administration of IL-1β. Adoptive transfer of the immunized spleen cells caused the complete regression of Meth-A tumors. These results suggest that intratumoral administration of IL-1β might induce cytotoxic cells in the left non-treated tumor of the double grafted tumor system and bring about the regression of metastatic tumors. On the other hand, recombinant tumor necrosis factor was effective only on the treated, right tumor, having no effect on the distant, left tumor in the double grafted tumor system. Recombinant interleukin-2 was effective on neither the right tumor nor the left tumor in this system. These results show that there are major differences of antitumor mechanism among cytokines.     

Antimetastatic effect of biological response modifiers in the "double grafted tumor system"

The antimetastatic effect of biological response modifiers (BRM) in a new experimental mouse model was studied. Intratumoral administration of BRMs (PSK, OK-432, interferon alpha A/D) strongly inhibited the growth of Meth-A solid tumors in male BALB/c mice and led to a complete regression of tumors and resistance to reinoculated tumors. Subsequently, the antimetastatic effect of BRMs was examined in the "double grafted tumor system," in which mice first received simultaneous intradermal inoculations of Meth-A in the right (10(6) cells) and left (2 X 10(5) cells) flanks and were then injected with BRMs in the right tumor on day 3. PSK and interferon (IFN) significantly inhibited the growth of the left (non-treated) tumor. This finding suggests that intratumoral BRM immunotherapy in one region has an effect on tumor growth in another region. Immunized spleen cells were taken from mice which had been cured by the intratumoral administration of BRMs and had rejected reinoculated tumors. One hour after intravenous injection of cyclophosphamide (2 mg/mouse), immunized spleen cells (2 X 10(7) cells/mouse) were injected into the Meth-A tumor on day 3. Adoptive transfers of PSK and IFN immunized spleen cells caused the complete regression of Meth-A tumors. These results suggest that the intratumoral administration of BRMs might induce cytotoxic cells in the left non-treated tumor of the "double grafted tumor system" and bring about the regression of metastatic tumors.     

Antitumor effect of a novel antitumor compound, NC-190, in the double grafted tumor system

The antitumor effect of NC-190, N-beta-dimethylaminoethyl-9-carboxy-5-hydroxy-10-methoxybenzo [alpha] phenazine-6-carboxamide sodium salt, with a new experimental mouse model was studied. Intratumoral administration of NC-190 strongly inhibited the growth of Meth-A solid tumors in male BALB/c mice and led to a complete tumor regression and also resistance to the reinoculated tumor. Subsequently, the anti-metastatic effect of NC-190 was examined in the double grafted tumor system, in which mice first received simultaneous intradermal inoculations of Meth-A in both right (10(6) cells) and left (2 X 10(5) cells) flanks and were then injected with 25 micrograms of NC-190 in the right tumor on days 3, 4 and 5. NC-190 inhibited the growth of not only the right but also the left, non-treated tumor. Immunized spleen cells were taken from mice which had been cured by the intratumoral administration of NC-190. Adoptive transfer of NC-190 immunized spleen cells caused a complete regression of Meth-A tumors. The effector cell activity was lost only after treatment with anti-Lyt-2 antibody. These results suggest that intratumoral administration of NC-190 might induce Lyt-2 positive cytotoxic cells in the spleen and the left, non-treated tumor. In BALB/c nude mice, NC-190 inhibited slightly the growth of the right tumor but did not that of the left tumor. Therefore, the antitumor activity of NC-190 in the double grafted tumor system was judged as associated with a sequential immune mechanism in which T cells may play an important role. TILs (tumor infiltrating lymphocytes) obtained from the left and right sides tumors treated with NC-190 were examined by Winn assay for their antitumor activity against Meth-A sarcoma in BALB/c mice. TILs obtained from neither sides inhibited the growth of admixed Meth-A cells. Different from immunopotentiators such PSK or IL-1, NC-190 enhanced neither concomitant immunity nor sinecomitant immunity.     

Antitumor Effector Mechanism at a Distant Site in the Double Grafted Tumor System of PSK, a Protein-bound Polysaccharide Preparation

The antitumor effect at a distant site of PSK, a Coriolus preparation, was analyzed with the double grafted tumor system in which BALB/c mice received simultaneous intradermal inoculations of Meth-A tumor in the right (10⁶ cells) and left (2 × 10⁵ cells) flanks and were then injected with PSK in the right-flank tumor on day 3. PSK inhibited the growth of not only the right but also the left (non-treated) tumor. Immunized spleen cells were taken from mice which had been cured by the intratumoral administration of 5 mg of PSK and were injected into the Meth-A tumor on day 3. Adoptive transfer of PSK immunized spleen cells caused the complete regression of Meth-A tumors. The effector cell activity was lost only after treatment with anti-Lyt-1 monoclonal antibody plus complement. Spleen cells and right and left regional lymph node cells prepared from PSK immunized mice were examined for Thy-1, Lyt-1, Lyt-2 and asialo GM1 phenotypes. The number of Lyt-1-positive lymphocytes increased in the right regional lymph nodes after intratumoral administration of PSK. A massive accumulation of macrophages and polymorphonuclear leukocytes was found in the right tumor and an infiltration of macrophages and Lyt-2-positive lymphocytes was found in the left (non-treated) tumor by immunohistochemical analyses. These results suggest that intratumoral administration of PSK induces Lyt-1-positive cells first in regional lymph nodes, then in the spleen, and subsequently induces macrophages and Lyt-2-positive cells in the left (non-treated) tumor, thus bringing about the regression of metastatic tumors.     

Antitumor effector mechanism of interleukin-1 beta at a distant site in the double grafted tumor system.

Recombinant human interleukin-1 beta (IL-1 beta) inhibited the growth of not only the right, but also the left non-treated tumor in a double grafted tumor system. Since the antitumor activity of IL-1 beta against the right and left tumors was not seen in nude mice, lymphocytes have a key role in the antitumor effect of intratumoral administration of IL-1 beta. TIL (tumor-infiltrating leukocytes) obtained from left and right side tumors treated with IL-1 beta were examined by Winn assay for their antitumor activity against Meth-A sarcoma in BALB/c mice. TIL from the right side clearly inhibited the growth of admixed Meth-A cells, but control TIL did not. Spleen cells and right and left regional lymph node cells prepared from IL-1-treated mice were examined for Lyt-1, Lyt-2 and L3T4 phenotypes. The number of Lyt-1-positive lymphocytes increased in the spleen and in the right regional lymph nodes after intratumoral administration of IL-1. Isolated tumor cells obtained from the right tumor treated with IL-1 beta and the left side tumor on day 6 were cultured in RPMI 1640 with 10% fetal calf serum for 24 h. The culture supernatants were harvested and tested for the presence of chemotactic activity for neutrophils or macrophages. Significant neutrophil chemotactic factor and macrophage chemotactic factor activities were detected in the culture media from IL-1-treated tumor tissues cultured for 24 h. Neither significant neutrophil nor macrophage chemotactic activity was detected in the media from untreated tumor tissues. These results suggest that intratumoral administration of IL-1 first induces neutrophils and macrophages in the right tumor, then Lyt-1-positive cells in the right regional lymph nodes and in the spleen, and subsequently induces macrophages in the left, non-treated tumor.     

Antitumor effect of Cepharanthin in the double grafted tumor system

The antitumor effect of Cepharanthin (CR) in a new experimental mouse model was studied. Intratumoral administration of CR strongly inhibited the growth of Meth-A solid tumors in male BALB/c mice and led to a complete regression of tumors and resistance to reinoculated tumor. Subsequently, the antimetastatic effect of CR was examined in the double grafted tumor system, in which mice first received simultaneous intradermal inoculations of Meth-A in both right (10(6) cells) and left (2 x 10(5) cells) flanks and were then injected with 0.5 mg of CR in the right tumor on days 3, 4 and 5. CR inhibited the growth of not only the right but also the left, nontreated tumor. Immunized spleen cells were taken from mice which had been cured with the intratumoral administration of CR. Adoptive transfer of CR immunized spleen cells caused the complete regression of Meth-A tumors. The effector cell activity was lost only after treatment with anti-Lyt-1 antibody. These results suggest that intratumoral administration of CR might induce Lyt-1 positive cytotoxic cells in the spleen and the left, non-treated tumor. In BALB/c nude mice, CR inhibited the growth of the right tumor but did not the left tumor. Therefore, the antitumor activity of CR on the left tumor in the double grafted tumor system is associated with a sequential immune mechanism in which T cells may play an important role. The antitumor effect of CR on the right tumor is direct cytotoxic effect. TILs (tumor infiltrating lymphocytes) obtained from left and right sides tumors treated with CR were examined by Winn assay for their antitumor activity against Meth-A sarcoma in BALB/c mice. TILs from both sides clearly inhibited the growth of admixed Meth-A cells and seems to play an important role on antitumor effect in both tumors.     

Antitumor effector mechanism at a distant site in the double grafted tumor system of PSK, a protein-bound polysaccharide preparation

The antitumor effect at a distant site of PSK, a Coriolus preparation, was analyzed with the double grafted tumor system in which BALB/c mice received simultaneous intradermal inoculations of Meth-A tumor in the right (10(6) cells) and left (2 x 10(5) cells) flanks and were then injected with PSK in the right-flank tumor on day 3. PSK inhibited the growth of not only the right but also the left (non-treated) tumor. Immunized spleen cells were taken from mice which had been cured by the intratumoral administration of 5 mg of PSK and were injected into the Meth-A tumor on day 3. Adoptive transfer of PSK immunized spleen cells caused the complete regression of Meth-A tumors. The effector cell activity was lost only after treatment with anti-Lyt-1 monoclonal antibody plus complement. Spleen cells and right and left regional lymph node cells prepared from PSK immunized mice were examined for Thy-1, Lyt-1, Lyt-2 and asialo GM1 phenotypes. The number of Lyt-1-positive lymphocytes increased in the right regional lymph nodes after intratumoral administration of PSK. A massive accumulation of macrophages and polymorphonuclear leukocytes was found in the right tumor and an infiltration of macrophages and Lyt-2-positive lymphocytes was found in the left (non-treated) tumor by immunohistochemical analyses. These results suggest that intratumoral administration of PSK induces Lyt-1-positive cells first in regional lymph nodes, then in the spleen, and subsequently induces macrophages and Lyt-2-positive cells in the left (non-treated) tumor, thus bringing about the regression of metastatic tumors.     

Antitumor effect of PSK: role of regional lymph nodes and enhancement of concomitant and sinecomitant immunity in the mouse

PSK, a Coriolus preparation, inhibited the growth of not only the right but also the left, non-treated tumor in a double grafted tumor system. In order to examine the role of lymph nodes and the spleen in the antitumor activity of PSK, regional (axillary and inguinal) lymph nodes and spleen were resected. Since in resected mice the antitumor activity of PSK against the right and left tumors was weakened, the regional lymph nodes and the spleen probably have a very important role in the antimetastatic effect of intratumoral administration of PSK. TIL (tumor-infiltrating lymphocytes) obtained from left and right side tumors treated with PSK were examined by Winn assay for their antitumor activity against Meth-A sarcoma in BALB/c mice. TIL from both sides clearly inhibited the growth of admixed Meth-A cells, but control TIL did not. A primary growth of Meth-A sarcoma inoculated into the right flank resulted in the generation of concomitant immunity to the growth of a second graft of the same tumor cells in the left flank. A significant inhibitory effect on the proliferation of the tumor cells inoculated secondarily was shown in mice bearing a primary right tumor that had previously been inoculated with PSK 3 times. After surgical excision of the primary tumor on day 6, daily oral administration of PSK significantly inhibited the growth of the secondary tumor inoculated on day 21, that is, PSK treatment also enhanced sinecomitant immunity. These observations suggest that presurgical intratumoral injection and postoperative oral administration of PSK are highly effective in eradicating metastatic tumors.     

Antitumor Effect of PSK: Role of Regional Lymph Nodes and Enhancement of Concomitant and Sinecomitant Immunity in the Mouse

PSK, a Coriolus preparation, inhibited the growth of not only the right but also the left, non-treated tumor in a double grafted tumor system. In order to examine the role of lymph nodes and the spleen in the antitumor activity of PSK, regional (axillary and inguinal) lymph nodes and spleen were resected. Since in resected mice the antitumor activity of PSK against the right and left tumors was weakened, the regional lymph nodes and the spleen probably have a very important role in the antimetastatic effect of intratumoral administration of PSK. TIL (tumor-infiltrating lymphocytes) obtained from left and right side tumors treated with PSK were examined by Winn assay for their antitumor activity against Meth-A sarcoma in BALB/c mice. TIL from both sides clearly inhibited the growth of admixed Meth-A cells, but control TIL did not. A primary growth of Meth-A sarcoma inoculated into the right flank resulted in the generation of concomitant immunity to the growth of a second graft of the same tumor cells in the left flank. A significant inhibitory effect on the proliferation of the tumor cells inoculated secondarily was shown in mice bearing a primary right tumor that had previously been inoculated with PSK 3 times. After surgical excision of the primary tumor on day 6, daily oral administration of PSK significantly inhibited the growth of the secondary tumor inoculated on day 21, that is, PSK treatment also enhanced sinecomitant immunity. These observations suggest that presurgical intratumoral injection and postoperative oral administration of PSK are highly effective in eradicating metastatic tumors.     

Antitumor Effector Mechanism of Interleukin-lβ at a Distant Site in the Double Grafted Tumor System

Recombinant human interleukin-lβ (IL-lβ) Inhibited the growth of not only the right, but also the left non-treated tumor in a double grafted tumor system. Since the antitumor activity of IL-lβ against the right and left tumors was not seen in nude mice, lymphocytes have a key role in the antitumor effect of intratumoral administration of IL-lβ. TIL (tumor-infiltrating leukocytes) obtained from left and right side tumors treated with IL-1β were examined by Winn assay for their antitumor activity against Meth-A sarcoma in BALB/c mice. TIL from the right side clearly inhibited the growth of admixed Meth-A cells, but control TIL did not. Spleen cells and right and left regional lymph node cells prepared from IL-1-treated mice were examined for Lyt-1, Lyt-2 and L3T4 phenotypes. The number of Lyt-1-positive lymphocytes increased in the spleen and in the right regional lymph nodes after intratumoral administration of IL-1. Isolated tumor cells obtained from the right tumor treated with IL-lβ and the left side tumor on day 6 were cultured in RPMI1640 with 10% fetal calf serum for 24 h. The culture supernatants were harvested and tested for the presence of chemotactic activity for neutrophils or macrophages. Significant neutrophil chemotactic factor and macrophage chemotactic factor activities were detected in the culture media from IL-1-treated tumor tissues cultured for 24 h. Neither significant neutrophil nor macrophage chemotactic activity was detected in the media from untreated tumor tissues. These results suggest that intratumoral administration of IL-1 first induces neutrophils and macrophages in the right tumor, then Lyt-1-positive cells in the right regional lymph nodes and in the spleen, and subsequently induces macrophages in the left, non-treated tumor.     

Antitumor Effector mechanism of plant alkaloid preparation, cepharanthin, by intratumoral administration

The antitumor effect of biscoclaurine alkaloids, at a distant site was examined in the double grafted tumor system, in which mice received simultaneous intradermal inoculations of Meth-A in both right (10(6) cells) and left (2 x 10(5) cells) flanks and were then injected with 0.5 mg of CR in the right tumor on days 3, 4 and 5. CR inhibited the growth of not only the right but also the left, non-treated tumor. In order to examine the role of lymph nodes in the antitumor activity of CR, regional (axillary and inguinal) lymph nodes were resected. Since in resected mice the antitumor activity of CR against the left tumor was weakened, the regional lymph nodes have a very important role in the antitumor effect of intratumoral administration of CR at a distant site. Spleen cells prepared from CR treated mice were examined for Lyt-1, Lyt-2 and L3T4 phenotypes. The number of Lyt-1 positive lymphocytes increased in the spleen after intratumoral administration of CR. Isolated tumor cells obtained from the right tumor treated with CR and the left side tumor on day 6 were cultured for 24 h. The culture supernatants were harvested and tested for the presence of chemotactic activity for neutrophil or macrophage. Significant neutrophil chemotactic factor (NCF) activity was detected in the culture media from CR-treated right tumor tissue, and macrophage chemotactic factor (MCF) activity was detected in the culture media from left tumor tissue. CR-induced NCF was partially neutralized by treatment with anti-human IL-8 IgG, and might be murine IL-8 factor. These results suggest that intratumoral administration of CR first induces neutrophils in the right tumor, then Lyt-1 positive cells in the spleen, and subsequently induces cytotoxic macrophages in the left, non-treated tumor, thus bringing about the regression of distant tumors.     

Antitumor effect of PSK at a distant site: inductions of interleukin-8-like factor and macrophage chemotactic factor in murine tumor

The antitumor effect of PSK, a Coriolus preparation, at a distant site was analyzed with the use of a double grafted tumor system in which male BALB/c mice received simultaneous intradermal inoculations of Meth-A tumor in the right (10(6) cells) and the left (2 x 10(5) cells) flanks and were then injected with PSK in the right tumor on the third day thereafter. The antitumor effect of intratumoral administration of PSK in the right tumor on days 3, 4 and 5 was compared with the effect of surgical resection of the right tumor on day 5. Three out of 8 mice given PSK intratumorally became tumor-free whereas no mouse tumor-free in the left flank was found among the surgically resected mice. As regards sinecomitant immunity, tumor inoculation into the right flank followed by intra-tumoral administration of PSK on days 3 and 5 and surgical excision of the primary tumor on day 6 resulted in complete rejection of a tumor challenge in the left flank on day 21. The combination of presurgical intratumoral injections of PSK (more than 2 times) and postoperative oral administration of PSK appeared to be most effective in eradicating secondary tumors. Isolated TILs (tumor-infiltrating lymphocytes), obtained from the right tumor (treated with PSK) and the left tumor on day 10 in the double grafted tumor system were cultured in RPMI1640 with 10% fetal calf serum for 24 h. The culture supernatants were harvested and tested for the presence of chemotactic activity for neutrophils or macrophages. Significant neutrophil chemotactic factor (NCF) and macrophage chemotactic factor (MCF) activities were detected in the culture media from PSK-treated TILs that had been cultured for 24 h. Neither significant neutrophil nor macrophage chemotactic activity was detected in the media from untreated TILs. NCF and MCF activities were also detected in the culture supernatant from PSK-treated tumor tissue on day 6. PSK-induced NCF in the murine tumor was neutralized by treatment with anti-human IL-8 IgG, and might be murine IL-8-like factor. Therefore, neutrophil and macrophage infiltrations of tumors following intratumoral injections of PSK are probably mediated by inductions of IL-8-like factor and MCF.     

Antitumor Effect of PSK at a Distant Site: Inductions of Interleukin-8-like Factor and Macrophage Chemotactic Factor in Murine Tumor

The antitumor effect of PSK, a Coriolus preparation, at a distant site was analyzed with the use of a double grafted tumor system in which male BALB/c mice received simultaneous intradermal inoculations of Meth-A tumor in the right (10⁶ cells) and the left (2 × 10⁵ cells) flanks and were then injected with PSK in the right tumor on the third day thereafter. The antitumor effect of intratumoral administration of PSK in the right tumor on days 3, 4 and 5 was compared with the effect of surgical resection of the right tumor on day 5. Three out of 8 mice given PSK intratumorally became tumor-free whereas no mouse tumor-free in the left flank was found among the surgically resected mice. As regards sinecomitant immunity, tumor inoculation into the right flank followed by intratumoral administration of PSK on days 3 and 5 and surgical excision of the primary tumor on day 6 resulted in complete rejection of a tumor challenge in the left flank on day 21. The combination of presurgical intratumoral injections of PSK (more than 2 times) and postoperative oral administration of PSK appeared to be most effective in eradicating secondary tumors. Isolated TILs (tumor-infiltrating lymphocytes), obtained from the right tumor (treated with PSK) and the left tumor on day 10 in the double grafted tumor system were cultured in RPMI1640 with 10% fetal calf serum for 24 h. The culture supernatants were harvested and tested for the presence of chemotactic activity for neutrophils or macrophages. Significant neutrophil chemotactic factor (NCF) and macrophage chemotactic factor (MCF) activities were detected in the culture media from PSK-treated TILs that had been cultured for 24 h. Neither significant neutrophil nor macrophage chemotactic activity was detected in the media from untreated TILs. NCF and MCF activities were also detected in the culture supernatant from PSK-treated tumor tissue on day 6. PSK-induced NCF in the murine tumor was neutralized by treatment with anti-human IL-8 IgG, and might be murine IL-8-like factor. Therefore, neutrophil and macrophage infiltrations of tumors following intratumoral injections of PSK are probably mediated by inductions of IL-8-like factor and MCF.     

Antitumor Effect of Intratumoral Administration of Biological Response Modifiers: Induction of Immunosuppressive Acidic Protein, a Type of α1-Acid Glycoprotein, in Mice

The antitumor effects of biological response modifiers (BRMs) in an experimental mouse model, the "double grafted tumor system" were analyzed. Male BALB/c mice received simultaneous inoculations of Metn-A fibrosarcoma cells on the right flank (10⁶ cells) and left flank (2 × 10⁵ cells) on day 0, and BRMs were injected intratumorally into the right tumor on days 3, 4 and 5. PSK (a protein-bound polysaccharide preparation), interleuldn-1 (IL-1) and cepharanthin (R) cured not only the right, but also the left, non-treated tumor in a double grafted tumor system. OK-432 (a Streptococcus preparation) and BCG and tumor necrosis factor (TNF) cured the right tumor and inhibited the growth of the left tumor. Lentinan (a polysaccharide preparation) and IL-6 inhibited neither the right nor the left tumor. Immunosuppressive acidic protein (IAP) in serum was increased transiently soon after intradermal injection of PSK, CR, OK-432 and TNF in BALB/c mice. Lentinan, however, did not induce IAP. IAP in serum was gradually increased after intradermal inoculation of Meth-A tumor in BALB/c mice. The biochemical difference between PSK-induced IAP (early, inflammatory IAP) and Meth-A-induced IAP (late, tumor-induced IAP) was investigated by crossed affinity immunoelectrophoresis with concanavalin A. IAP of murine serum was separated into 4 peaks. IAP in normal mouse was rich in high-mannose type sugar chain (Peak 3) and contained no hybrid-type sugar chain (Peak 4), which was present in inflammatory and tumor-induced IAP. Inflammatory IAP was rich in biantennary sugar chain (Peak 2) and tumor-induced IAP was rich in tri-tetraantennary sugar chain (Peak 1).     

Antitumor effect of PSK at a distant site: tumor-specific immunity and combination with other chemotherapeutic agents.

The antitumor effect of PSK, a Coriolus preparation, was analyzed with the double grafted tumor system in which BALB/c mice received intradermal inoculations of syngeneic Meth-A fibrosarcoma in the right (primary tumor, 10(6) cells) and left (distant tumor, 2 x 10(5) cells) flanks. Intratumoral administration of PSK significantly inhibited the growth of not only the right but also the left tumor. PSK also inhibited the growth of a methylcholanthrene-induced fibrosarcoma BAMC-1, and a methylurethane-induced adenocarcinoma Colon 26 in the double grafted tumor system of syngeneic BALB/c mice. However, when the left distant tumor was different from the right Meth-A tumor, the intratumoral administration of PSK in the right tumor was unable to inhibit the growth of the left BAMC-1 or RL male-1 tumor. The PSK-induced immunity, therefore, is tumor-specific and T lymphocytes may play an important role in antitumor memory function. The enhancement of concomitant immunity by PSK treatment was completely impaired by previous intravenous administration of an alkylating agent, cyclophosphamide (CY). The enhancement of sinecomitant immunity by PSK treatment was also impaired by previous CY intravenous administration. The antitumor effect of PSK was suppressed by previous intravenous administration of another alkylating agent, ACNU. It is possible that alkylating agents suppress the function of effector T cells and granulocytes which are very important for the antitumor immune cascade reaction due to PSK treatment. On the other hand, the antitumor effect of PSK was enhanced by previous intravenous administration of an anti-metabolite, 5-fluorouracil.     

Antitumor Effect of PSK at a Distant Site: Tumor-specific Immunity and Combination with Other Chemotherapeutic Agents

The antitumor effect of PSK, a Coriolus preparation, was analyzed with the double grafted tumor system in which BALB/c mice received intradermal inoculations of syngeneic Meth-A fibrosarcoma in the right (primary tumor, 10⁵ cells) and left (distant tumor, 2 × 10⁵ cells) flanks. Intratumoral administration of PSK significantly inhibited the growth of not only the right but also the left tumor. PSK also inhibited the growth of a methylcholanthrene-induced fibrosarcoma BAMC-1, and a methylurethane-induced adenocarcinoma Colon 26 in the double grafted tumor system of syngeneic BALB/c mice. However, when the left distant tumor was different from the right Meth-A tumor, the intratumoral administration of PSK in the right tumor was unable to inhibit the growth of the left' BAMC-1 or RL♂ -1 tumor. The PSK-induced immunity, therefore, is tumor-specific and T lymphocytes may play an important role in antitumor memory function. The enhancement of concomitant immunity by PSK treatment was completely impaired by previous intravenous administration of an alkylating agent, cyclophosphamide (CY). The enhancement of sinecomitant immunity by PSK treatment was also impaired by previous CY intravenous administration. The antitumor effect of PSK was suppressed by previous intravenous administration of another alkylating agent, ACNU. It is possible that alkylating agents suppress the function of effector T cells and granulocytes which are very important for the antitumor immune cascade reaction due to PSK treatment. On the other hand, the antitumor effect of PSK was enhanced by previous intravenous administration of an anti-metabolite, 5-fluorouraeil.     

Intratumoral therapy with IL13-PE38 results in effective CTL-mediated suppression of IL-13Ralpha2-expressing contralateral tumors.

PURPOSE: IL13-PE38, a targeted cytotoxin comprised of interleukin 13 (IL-13) and a mutated form of Pseudomonas exotoxin, induces specific killing of tumor cells expressing abundant levels of the IL-13Ralpha2 chain. We hypothesized that tumor cells killed by the cytotoxin may release antigens and/or apoptotic bodies when cells are dying, which then induce adoptive immunity, and that the PE38 portion of IL13-PE38 may act as a stimulant for the induction of a CTL response. EXPERIMENTAL DESIGN: To test this hypothesis, we established D5 melanoma tumors with or without expression of the IL-13Ralpha2 chain in both flanks of C57BL/6 mice, and then IL13-PE38 was injected in the right flank tumors only. Results and CONCLUSIONS: When animals with IL-13Ralpha2-expressing D5 tumor (right) were injected with IL13-PE38, right flank tumors expressing the IL-13Ralpha2 chain not only showed dramatic regression but contralateral tumors (left flank) also showed tumor regression. Cell depletion experiments in tumor-bearing animals indicated that both CD8(+) and CD4(+) T cells contribute to the regression of contralateral tumors through CTL activation in the periphery and cellular infiltration into tumors. In addition, intratumoral treatment into s.c. tumors of mice bearing metastatic lung tumors with IL13-PE38 showed not only the reduction of treated s.c. tumor but also the reduction of lung metastasis. Thus, IL13-PE38 mediates an antitumor effect not only directly but also indirectly by inducing a host CD8(+) T cell immune response. Accordingly, targeted cytotoxins may be used to treat local disease even if they cannot be administered systemically, and yet may still induce a reasonable systemic antitumor response.     

Effect of interleukin (IL)-4 cytotoxin on breast tumor growth after in vivo gene transfer of IL-4 receptor alpha chain.

Although human breast cancer cells express interleukin-4 receptors (IL-4Rs), a recombinant fusion protein, IL-4 cytotoxin, did not mediate desirable antitumor activity in tumor models of breast cancer. Recent studies have identified that a primary IL-4 binding protein, IL-4Ralpha chain, is internalized after binding to IL-4 in cancer cells. The consequent expression of high-level IL-4Ralpha in tumor cells sensitizes them to the cytotoxic effect of IL-4 cytotoxin in vitro. To assess whether overexpression of IL-4Ralpha chain in vivo by plasmid-mediated gene transfer can enhance antitumor activity of IL-4 cytotoxin in mouse models of breast tumor, we injected MDA-MB-231 human breast cancer cells in both flanks of athymic nude mice. Animals then received three intratumoral (i.t.) injections of either IL-4Ralpha encoding vector (left flank) or vector only (right flank) mixed with liposome followed by IL-4 cytotoxin administration. Both i.p. and i.t. administration of IL-4 cytotoxin profoundly reduced the growth of IL-4Ralpha plasmid-injected MDA-MB-231 tumors, compared with control. Innate immune cells, including macrophages and neutrophils, were found to infiltrate at the regressing tumor site. This study provides proof of principle that i.t. IL-4Ralpha plasmid injection followed by systemic or i.t. IL-4 cytotoxin administration may be a useful strategy for the treatment of breast cancer.     

Inhibition of tumor growth by the intralesional administration of interleukin-3 into mice implanted with solid tumors.

The antitumor activity of three interleukin-3 (IL-3) preparations administered intralesionally into mice bearing syngeneic solid tumors was investigated. IL-3 preparations used in this study included S-IL-3, which was isolated from the culture fluid of murine inguinal lymph node cells stimulated with an arabinomannan extracted from Mycobacterium tuberculosis (SSM), the culture fluid of WEHI-3 cells (W-IL-3) and recombinant IL-3 (rIL-3). When a 1,500 U/kg dose of S-IL-3 or W-IL-3 was injected intralesionally into BALB/c mice bearing Meth-A solid tumors three time per week beginning 3 days after tumor inoculation, tumor growth was inhibited by 60% or 74% at 24 days after tumor inoculation, respectively. In these experiments, 1 unit of IL-3 activity was determined to be the concentration that induced 50% of maximal proliferation of an IL-3 dependent cell line (FCD-P2 cells). The administration of this dose of rIL-3 inhibited tumor growth by 34%. When these three preparations of IL-3 were pretreated with anti-IL-3 monoclonal antibody in vitro, the antitumor activity, as well as their growth promoting activity on FDC-P2 cells, was eliminated. Since direct cytotoxic activities of these IL-3 preparations against cultured Meth-A tumor cells in vitro have not been demonstrated, these results suggest that their antitumor activities might be expressed through interactions between tumor cells and antitumor effector cells which were stimulated by the intralesional administration of the IL-3 preparations.     

Differences in the immunostimulatory activity and antitumor therapeutic effect of OK-432 and N-CWS

Immunobiological activities of OK-432 and N-CWS were studied in BALB/c-Meth-A and C3H-MH 134 systems. Peritoneal exudate cells obtained from mice injected i.p. with OK-432 or N-CWS showed stronger in vitro cytotoxicity against Meth-A and YAC-1 cells than did those from non-treated mice. Spleen cells from OK-432 or N-CWS-treated mice exhibited lower Con A mitogenic response in comparison with those from non-treated mice. When Meth-A or MH-134 tumor cells were admixed with OK-432 or N-CWS, the intradermally inoculated tumor cells showed no or slower growth than did those admixed with saline only. There were no significant differences in all the above mentioned activities between OK-432 and N-CWS, and there was no difference in the direct tumor inhibitory activity of the two agents in vitro. However, the administration of N-CWS had no therapeutic effect against i.p.-inoculated tumor cells while OK-432 showed a strong therapeutic effect by the same treatment schedule. Further studies are required to clarify the mechanisms responsible for the difference in the therapeutic effect of the two agents.