Double Grafted Tumor System Research Articles

Morphological Observations on Antitumor Activities of 70kDa Fragment in .ALPHA.-Subunit from Pronase-Treated Ovomucin in a Double Grafted Tumor System.

The antitumor activities of a 70 kDa highly glycosylated fragment (OVMα70F) in the α-subunit separated from pronase-treated hen egg white ovomucin were examined in a double grafted tumor system. BALB/c mice received simultaneous inoculations of Meth-A tumor cells in the right flank (1×106 cells) and left flank (2×105 cells). Solutions (250 μl) of OVMα70F (1000 μg/mouse/day) or physiological saline (PS) were injected into the right tumor on days 3, 4, and 5, and the mice were raised for 21 days. OVMα70F induced direct and almost complete inhibition of growth of the right (treated) tumor and acted indirectly and moderately on growth of the left (distant), compared with each control tumor. The microscopic observations of tumor tissues on day 21 showed that a greater portion of the OVMα70F-treated right tumor cells was necrotic, whereas a smaller portion of the left one was degenerative. In the tumor tissues of OVMα70F-treated mice neutrophils, macrophages and lymphocytes were found to have massively accumulated and the angiogenesis (the formation of new capillary blood vessels) was inhibited.

Antitumor Effects of Pronase-Treated Fragments, Glycopeptides, from Ovomucin in Hen Egg White in a Double Grafted Tumor System

Antitumor effects of fragments (220 and 120 kDa, highly glycosylated peptides) separated from Pronase-treated hen egg white ovomucin were analyzed in a double grafted tumor system. BALB/c mice received simultaneous inoculations of Meth-A fibrosarcoma cells on the right flank (1 × 106 cells) and left flank (2 × 105 cells) on day 0. The two fragments (100 μg/mouse/day) were injected into the right tumor on days 3, 4, and 5, and mice were raised for 21 days. Both fragments cured directly and entirely the right (treated) tumor and inhibited indirectly and slightly the growth of the left (distant) one. Examinations of desialylated 120 kDa fragment indicated that the sialic acid residues in the 120 kDa fragment are not necessarily essential for direct antitumor activity but might be indispensable for regression of distant tumors. The noninhibitory activities in a single tumor system, in which mice received intradermal inoculation of tumor cells only in the left flank, and the increase of immunosuppressive acid protein in serum suggested the slight activation of the immune system. Keywords: Ovomucin; antitumor effect; glycopeptide

Selective tumoricidal effect of soluble proteoglucan extracted from the basidiomycete, Agaricus blazei Murill, mediated via natural killer cell activation and apoptosis.

We have isolated a novel type of natural tumoricidal product from the basidiomycete strain, Agaricus blazei Murill. Using the double-grafted tumor system in Balb/c mice, treatment of the primary tumor with an acid-treated fraction (ATF) obtained from the fruit bodies resulted in infiltration of the distant tumor by natural killer (NK) cells with marked tumoricidal activity. As shown by electrophoresis and DNA fragmentation assay, the ATF also directly inhibited tumor cell growth in vitro by inducing apoptotic processing; this apoptotic effect was also demonstrated by increased expression of the Apo2.7 antigen on the mitochondrial membranes of tumor cells, as shown by flow-cytometric analysis. The ATF had no effect on normal mouse splenic or interleukin-2-treated splenic mononuclear cells, indicating that it is selectively cytotoxic for the tumor cells. Cell-cycle analysis demonstrated that ATF induced the loss of S phase in MethA tumor cells, but did not affect normal splenic mononuclear cells, which were mainly in the G0G1 phase. Various chromatofocussing purification steps and NMR analysis showed the tumoricidal activity to be chiefly present in fractions containing (1-->4)-alpha-D-glucan and (1-->6)-beta-D-glucan, present in a ratio of approximately 1:2 in the ATF (molecular mass 170 kDa), while the final purified fraction, HM3-G (molecular mass 380 kDa), with the highest tumoricidal activity, consisted of more than 90% glucose, the main component being (1-->4)-alpha-D-glucan with (1-->6)-beta branching, in the ratio of approximately 4:1.

Antitumor effect of a peptide-glucan preparation extracted from Agaricus blazei in a double-grafted tumor system in mice.

The antitumor effect of extracts obtained from the fruit body of Agaricus blazei Murill was examined in a double-grafted tumor system, in which BALB/c mice received simultaneous intradermal injections of Meth-A tumor cells in both the right (10(6) cells) and left flank (2 x 10(5) cells), and were then injected with 5 mg of extracts of A. blazei in the right tumor on days 3, 4 and 5. Intratumoral administration of ethanol-soluble (Fraction 1), water-ethanol-soluble (Fraction 2), ammonium oxalate-soluble (Fraction 3) and ammonium oxalate-insoluble (Fraction 4) fractions resulted in inhibition of tumor growth, with Fraction 3 showing the most tumoricidal activity, producing regression of the right tumor and inhibition of growth of the left, non-injected tumor. The maximum effect was obtained using 0.5 mg of Fraction 3 and this amount was used in subsequent experiments. The antitumor effect of intratumorally administered Fraction 3 was enhanced by oral ad lib administration of feed containing 0.083% of Fraction 3. When immunized spleen cells from mice that had been cured by intratumoral administration of 0.5 mg of Fraction 3 were directly injected (2 x 10(7) cells/mouse) into the Meth-A tumor, tumor growth was inhibited. The tumor cells on day 7 from the Fraction 3-treated right tumor and from the left tumor were cultured for 24 h and their culture supernatants were assayed for neutrophil or macrophage chemotactic activity. Significant macrophage chemotactic factor activity was detected in the culture media from the left tumor tissue. Serum levels of immunosuppressive acidic protein (IAP), produced by activated macrophages and neutrophils, increased transiently soon after intradermal injection of 0.5 mg of Fraction 3. These results suggest that regression of the left non-injected tumor was due to an immune reaction, involving induction of cytotoxic cells in the spleen, and the release of chemotactic factors in the distant tumor.

Augmentation of tumor immunity in mice by intralesional injection of vitamin A.

We investigated the antitumor effect of vitamin A(VA) using the double grafted tumor technique to examine whether VA administered into a primary tumor (intralesionally or i.l.) accelerates antitumor immune reactions so that growth of the secondary tumor may be more effectively inhibited than by other systemic administration routes. In the double grafted tumor system, where BALB/c mice were inoculated with MethA fibrosarcoma cells into the right inguinal region (1 x 10[6] cells) on day 0 and later into the left (3 x 10[6] cells) on day 10, the injection of VA at a dose of 1000 IU/mouse i.l., s.c., i.p., and i.v. on days 3 through 7 inhibited the growth of the secondary tumor to the same extent, while VA at the i.l. dose of 100 IU/mouse into the primary tumor inhibited more effectively than by any other administration route. VA did not inhibit the secondary MethA growth in BALB/c (nu/nu) mice. The spleen cells taken from VA-treated tumor-bearing mice prevented the growth of MethA tumors in naive BALB/c mice when given as a mixture with the MethA inoculum (the Winn assay). The delayed-type hypersensitivity (DTH) response to methylated bovine serum albumin (MBSA) antigen was augmented when VA (1000 IU) was injected at the site of the antigen injection. These results suggest that the direct interaction of VA with the tumor cells may be necessary for the tumor immunity-potentiating effect of VA, and that T-lymphocyte-mediated tumor immunity is involved in the anti-tumor effect of VA. The antitumor mechanism of VA seems to involve retinoid receptors, because the benzoic acid derivative Am80, which has been reported to exert retinoidal activity by binding to specific retinoid receptors, also showed activity.

Study on the mechanism of immunopotentiating antitumor effect of 6-MPG, a water-soluble derivative of 6-mercaptopurine

We investigated possible mechanisms of the antitumor action of γ-(9H-purine-6-yl) thiomethyl l-glutamate (6-MPG), a water-soluble derivative of 6-MP. In the double grafted tumor system, BALB/c mice were inoculated intradermally with 106 cells of MethA fibrosarcoma at the right inguinal region on day 0 (the primary tumor) and later with 3×106 cells at the left on day 10 (the secondary tumor). Intraperitoneal administration of 6-MPG at a dose of 100 mg/kg/day from day 3 through 7 completely prevented growth of the secondary tumor. 6-MPG showed no effect on growth of colon 26 adenocarcinoma cells inoculated in place of the secondary MethA cells (antigen specificity). 6-MPG did not inhibit the secondary MethA growth in the BALB/c (nu/nu) mouse. The inhibitory effect of 6-MPG on the secondary tumor growth was diminished by prior treatment of the primed animals with cyclosporin A and anti-Thy antibody. Spleen cells from the tumor-bearing mice treated with 6-MPG showed a tumor-neutralizing activity (Winn assay). Treatment of the spleen cells with anti-CD8 antibody plus complement diminished the tumor-neutralizing effect but that with anti-CD4 antibody plus complement did not, indicating that CD8-positive cells are responsible for potentiation of the tumor immunity. These results suggest that the antitumor effect of 6-MPG against the secondary tumor is elicited by augmenting tumor specific T-cell production.

Augmentation of tumor immunity by 6-mpg, a water-soluble derivative of 6-mercaptopurine, in mice

Inhibitory effects on some immunological responses and MethA fibrosarcoma growth in the double grafted tumor system in mice were compared between 6-mercaptopurine (6-MP) and its novel water-soluble derivative, γ-(9 H-purine-6-yl)thiomethyl l-glutamate (6-MPG). The dose-dependent inhibitory effects by 6-MPG on the hemagglutinin response to SRBC, DTH reaction to MBSA, contact sensitivity to oxazolone, GVH response and growth of the primary tumor were 3-10 times weaker than those by 6-MP, probably reflecting the difference in their cytotoxicities as antimetabolites. However, the two drugs were nearly equipotent in producing inhibition of the secondary tumor growth, which is a host-mediated immunological response to tumor antigen as shown by its dependency on the primary inoculation with 1 × 10 4 or more MethA cells and by the production of anti-tumor splenocytes in tumor-bearing animals (the Winn assay). Thus, 6-MPG may point to the direction of derivatization towards anti-tumor immunopotentiators with an improved therapeutic index.

Augmentation of tumor immunity by 6-mercaptopurine (6-MP) and its analogs in the double grafted tumor system in mice.

We investigated the antitumor effect of 6-mercaptopurine (6-MP) and its analogs using the double grafted tumor technique. BALB/c mice were inoculated intradermally with MethA fibrosarcoma cells at the right inguinal region on day 0 (the primary tumor) and at the left on day 10 (the secondary tumor). Intraperitoneal or intra-lesional administration of 6-MP, 6-mercaptopurine riboside (6-MP-r) and 6-mercaptopurine riboside triacetate (6-MPRTA) from day 3 to 7 dose-dependently inhibited growth of the secondary tumor. Without the primary inoculation, 6-MP showed no effect on growth of the tumor inoculated on day 10, indicating that the antitumor effect of 6-MP could not be attributable to its direct antimetabolic or tumoricidal action only, and that the primary tumor inoculation is necessary for these compounds to inhibits growth of the challenging tumor. 6-MP did not inhibit the secondary MethA growth in the BALB/c (nu/nu) mouse. Both CD4+ and CD8+ T cells increased in the spleen of mice treated with 6-MP. Meanwhile, delayed-type hypersensitivity (DTH) reaction to the methylated bovine serum albumin (MBSA) antigen at the footpad was not augmented but inhibited by 6-MP-r and 6-MPRTA in both normal and tumor-bearing mice. Thus, the immunomodulatory activity of 6-MP could be observed in two opposite directions, augmentation of tumor immunity and inhibition of DTH to MBSA. This indicates that the immune mechanism and/or the type of effector cells induced in these two cell-mediated immune systems are different from each other.

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).

Relationship between the Tissue Distribution and Antitumor Activity of Highly Branched (1.RAR.3)-.BETA.-D-Glucan, SSG.

Distribution of 3H-labeled (1-->3)-beta-D-glucan([3H]SSG) obtained from the culture filtrate of Sclerotinia sclerotiorum IFO 9395, in various tissues in tumor-bearing mice was examined. [3H]SSG administered intra-peritoneally was mainly detected in liver, spleen, kidney and tumor masses. In contrast to i.p. administration, intra-lesionally administered [3H]SSG was not released from the tumor. Similarly, in a double grafted tumor system, [3H]SSG was located in the administered tumor and not distributed in the distant site tumor, in spite of the fact that significant antitumor effect was shown in both tumor sites in this system. Winn assay confirmed the activation of the systemic antitumor immunity. These results suggested that the distribution of glucans would be one important factor in determining their antitumor effects. However, this would not always be necessary if systemic immunity could be induced.

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.

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 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.

The “double grafted tumor system”, proposed to find effector cells in the analyses of antitumor effect of BRMs

The antitumor effects of three biological response modifiers (BRMs; PSK, IFN alpha A/D and OK432) and two chemotherapeutics (Mitomycin C and Neocarzinostatin) in a new experimental mouse model, the "double grafted tumor system," were evaluated. BALB/c mice received simultaneous inoculations of Meth A fibrosarcoma cells on right flank (1 x 10(6) cells) and left flank (2 x 10(5) cells) on day 0, and drugs were given intratumorally into the right-flank tumor on day 3. The growth of the left-flank tumor was the real target for the evaluation of a given drug after 21 days. All tested five agents successfully cured the drug-injected right tumor with a pre-determined optimum dose. In addition, PSK, OK432, IFN alpha A/D and MMC among the five, inhibited the left-flank tumor, whereas no inhibition was observed when treated with NCS. To understand the mechanism by which the antitumor effect of the above four agents is able to influence the growth of tumor on the other side, tumor cells (2 x 10(5) cells) inoculated only into the left flank were treated with drugs given subcutaneously to the right flank (single tumor system). Among the four, MMC exhibited an effect similar to that obtained in the double tumor system, and IFN alpha A/D showed a less pronounced but still definite antitumor effect. However, PSK and OK432 failed to express anti-tumor effect in the single tumor system. These results obtained with PSK, OK432 and IFN alpha A/D suggest that the effect of the drug on the left-tumor may be mediated by certain effector cells, which are specifically induced by injection of the drug, in the right-tumor tissues. When effector cell analysis was conducted with spleen cells obtained after PSK treatment by means of intratumoral adoptive transfer into 3-day Meth A bearing recipients, these cells were shown to be Lyt-1+2(-)-T and L3T4(+)-T cell.

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.

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 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.

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.