Intra-lesional injection of immunostimulants in bilateral rat tumors

Rats grafted with either two or six fragments of isogenic methylcholanthrene-induced transplantable fibrosarcomas were treated IT¹ with living BCG or killed C. parvum or a mixture of both immunostimulants. Various tumor combinations and therapeutic agent dosages were compared. When two fragments of the same tumor McFiFi2 (S) were grafted simultaneously, IT treatment of one of these with 2 mg of BCG induced cure of both in 50% of the animals, but IT injection of 2 × 10⁹ C. parvum was completely without effect in this situation. The prognosis was however, improved when the dose of immunostimulant was increased. The best results were obtained when each individual tumor in rats with two or six simultaneous identical grafts were treated with a mixture of BCG and C. parvum. The combination of IT immunostimulant treatment and surgical excision of the treated lesion demonstrated a persistence of the curative effect on the remote untreated tumor. Double grafting with isologous non-identical tumors revealed the influence of tumor burden and of the specificity of anti-tumor action of the treatment. The distant specific regression obtained in this system implies a specific immunological mechanism mediated by effector cells and/or antibodies which can circulate, identify the target cell and destroy it. This is in accordance with morphological observations. The intimate contact between BCG and the growing structured tumor is necessary for the therapeutic phenomenon.     

Safety and efficacy of living BCG or BCG cell walls (CW) in the treatment of guinea pig hepatoma.

Guinea pigs with established intradermal tumors and microscopic lymph node metastases were treated by intralesional injection of graded doses of living BCG or BCG CW. The lowest dose of living BCG used produced a significant cure rate and no grossly evident toxicity. An intermediate and the highest dose of living BCG used cured some animals but others lost weight and a significant number died as a result of the treatment. Histologic examination of animals with significant weight loss showed fatty degeneration of the liver, granulomatous hepatitis and histiocytic infiltration of the spleen. None of the doses of BCG CW used was toxic and they were at least as effective as living BCG in intralesional treatment. In some experiments in which treatment was delayed it was found that the extent of disease required to render treatment ineffective was about the same for living BCG and for BCG CW.     

Tumor growth inhibition and potentiation of immunotherapy by indomethacin in mice.

Indomethacin was continuously administered in the drinking water of inbred C3H mice given grafts of syngeneic 3-methylcholanthrene-induced fibrosarcomas. A minor proportion of these animals died at the same time as the untreated controls, and others completely rejected their tumors; however, in most cases, the tumor growth rate was significantly slowed, and growth recommenced rapidly after drug withdrawal. This was the pattern for tumors either in their 10th to 14th transplant generation or only their third in vivo passage. Indomethacin exerted little prophylactic effect, in that it neither increased the minimal cell number required to initiate tumor growth nor significantly decreased the proportion of tumors established in drug-treated animals recieving tumor grafts. The injection of killed Corynebacterium parvum organisms into small, growing McC3-I tumors [intratumor (IT) route] caused the regression of most of these. In contrast, IT injection of BCG, ip injection of C. parvum, or IT injection of C. parvum into larger tumors had no effect. Oral administration of indomethacin enhanced BCG treatment and augmented the activity of C. parvum injected either systemically into animals with small tumors or IT into those with substantial tumor burdens. The duration of these effects was, however, often dependent on the continued administration of the drug.     

Combined intratumoral injection of bone marrow-derived dendritic cells and systemic chemotherapy to treat pre-existing murine tumors

Dendritic cells (DCs) are attractive candidates for innovative cancer immunotherapy by virtue of their potential to function as professional antigen-presenting cells for initiating cellular immune responses. In this study, we evaluated a possible synergy of conventional chemotherapy together with intratumoral injection of syngeneic bone marrow-derived DCs for the treatment of preexisting tumors. Using murine CT26 colon adenocarcinoma cells (parental or modified to express beta-galactosidase as a model tumor antigen) to produce s.c. tumors in syngeneic BALB/c mice, the data demonstrate that direct injections of DCs at the tumor site result in partial eradication of established tumors. Strikingly, the addition of systemic chemotherapy (cyclophosphamide) combined with local intratumoral injection of DCs led to complete tumor regression in the treated animals. The tumor-free mice were able to resist a repeat challenge with the same tumor, suggesting that the animals had acquired long term antitumor immunity. Supporting evidence for the paradigm of systemic chemotherapy and intratumoral administration of DCs was obtained using melanoma B16 syngeneic tumor treated with Adriamycin plus DCs. These novel findings raise the possibility of using this potent strategy of combined intratumoral injections of DCs and systemic chemotherapy for cancer treatment.     

Cytotoxic effect of anti-Mr 67,000 protein immunotoxins on human tumors in a nude mouse model

We have studied the potential use of immunotoxins (ITs) for therapeutic treatment of human tumors in an experimental model of human neoplasia. We tested intact ricin IT for its antitumor activity against established tumors. CEM, a human T-cell leukemia line expressing an Mr 67,000 cell surface antigen, and Daudi, a human B-cell lymphoma line which does not express the antigen, were found to be consistently tumorigenic in nude mice. ITs were synthesized using T101, a high-affinity monoclonal antibody reacting with the Mr 67,000 protein determinant and intact ricin. We have shown for the first time that established CEM solid tumors in nude mice will regress following intratumoral injection of T101-ricin IT, while Daudi tumors will not. Selective activity of T101-ricin is dependent on systemic i.v. administration of lactose and local intratumoral injection of the T101-ricin IT with lactose. Intact ricin ITs require the presence of lactose to block native ricin binding and render them antigen specific when linked to monoclonal antibody. Killing of target was cell specific since (a) nonspecific (irrelevant) ITs did not cause the regression of CEM tumors, and (b) injection of large amounts of free T101 antibody prior to T101-ricin IT blocked antitumor activity. Selectivity was not absolute, since regression occurred in one of six animals given irrelevant IT, and blocking was observed in two of four mice. Intratumoral IT treatment with 1 or 2 micrograms of T101-ricin IT plus lactose was not harmful to mice in contrast to intratumoral ricin treatment, which killed all treated tumor-bearing mice at a dose of 0.3 micrograms. Without i.v. injection of lactose, intratumoral injection of T101-ricin IT was also effective in eliminating established tumors. However, this treatment did not result in the selective elimination of tumor, since Daudi tumors also regressed following T101-ricin IT treatment. IT, made with ricin A chain only (T101-A chain IT), was also tested against established CEM tumors. We found that high dosages of T101-A chain IT did not destroy CEM tumors when injected intratumorally, even in the presence of activating agents such as NH4Cl or the carboxylic ionophore X-537 A. In contrast, in vitro experiments demonstrated that T101-A chain IT plus activating agents had potent and selective cytotoxic effect against CEM cells. We conclude that ITs are specifically toxic to established tumors. Although selectivity is not absolute, ITs exhibit potential as a new class of antitumor reagents.     

Suppression and immunotherapy of the guinea pig line-10 hepatocarcinoma mediated by nonviable Staphylococcus aureus.

Suppression of growth of the line-10 guinea pig hepatocarcinoma was achieved after the simultaneous injection of line-10 cells and heat-killed Staphylococcus aureus. Growth of tumor was also suppressed when line-10 cells were injected alone, contralaterally, at the same time as the vaccine mixture. Immunity developed to subsequent line-10 cell challenges but not to other syngeneic tumors. Similar results were obtained with the use of protein A-rich or -deficient strains of S. aureus. Multiple intratumor injections of heat-killed S. aureus were therapeutically effective against 6-day tumors. The antitumor effects of nonviable S. aureus were similar in many ways to those of living BCG or bacterial products suspended in oil droplets.     

瘤体内直接注射白介素2质粒复合物治疗小鼠肝癌

目的研究瘤体内直接注射白介素2质粒/阳离子脂质体复合物治疗小鼠肝癌的效果.方法将小鼠白介素2表达质粒(VR1110)与阳离子脂质体(Transfectam)按适当比例混合而形成复合物(VR1110/Transfectam).通过瘤体内直接注射此复合物治疗小鼠肝癌模型,并和卡介苗(BCG)的治疗相比较.结果瘤体内注射VR1110/Transfectam后,可在肿瘤组织中检测到小鼠IL-2 mRNA的表达.VR1110/Transfectam治疗组以及VR111/Transfectam/BCG治疗组肿瘤生长较其它组明显减慢,均显著提高小鼠的生存率(R＜0.05),但二者相差不明显.结论瘤体内直接注射VR1110/Transfectam复合物对小鼠肝癌的治疗作用明显,效果优于卡介苗,且方法简单,适合临床应用.     

Immunotherapy of primary methylcholanthrene-induced mouse tumours by intratumoral BCG.

Tumours were induced s.c. in C3H/uip, SJL/uip, DBA/2 uip, C57BL/6 uip and BDF1 mice by different doses of methylcholanthrene (MCA) diluted in oil: 1 mg, 0.1 mg and 0.01 mg. In each mouse strain, tumour frequency showed a different decreasing pattern in relation to the decreasing dose of MCA. Tumour latent period (LP) increased between the 1mg and 0.1mg doses of MCA, but the 0.01mg dose induced tumours with a similar or shorter LP than those tumours induced by 1 mg. Half of the tumours were treated with two injections of intratumoral (IT) BCG. The strains of mice differed in their sensitivity to this treatment, but only tumours induced by 0.01 mg MCA were sensitive to IT BCG. The induction of tumours by MCA pellets gave similar results. After transplantation of the untreated tumours, very few were cured by BCG treatment. Analysis of the role of tumour LP, growth rate and immunogenicity favours a slow growth rate as the most important characteristic for BCG sensitivity of the primary tumour. The tumours induced by 0.01 mg MCA were less immunogenic than those induced by 1 mg MCA, but the difference was not significant. This finding permits us to exclude an important role for tumour immunogenicity in the sensitivity of the primary tumour to BCB.     

Antitumor activity of murine tumor necrosis factor (TNF) against transplanted murine tumors and heterotransplanted human tumors in nude mice

The antitumor activity of highly purified tumor necrosis factor (TNF) was tested against eight kinds of murine tumor and five kinds of human tumor heterotransplanted into nude mice. Mice were treated by intravenous or intratumoral injection of TNF, commencing when the tumors were well established. TNF showed an excellent curative effect against all kinds of murine and human tumors tested. Meth A sarcoma, Colon 26, Ehrlich, sarcoma 180, MM 46, MH 134, B16 melanoma, and Lewis lung tumors transplanted into mice underwent tumor necrosis and regression following a single injection of TNF. Sometimes a complete cure was observed in Meth A sarcoma, sarcoma 180, Ehrlich, and MM 46 tumors. Human cancers, SEKI, HMV-I, KATO-III, MKN 45, or KB, heterotransplanted into nude mice, also exhibited tumor necrosis and regression in size following several intratumoral injections of TNF. A great difference in curative effects of TNF was observed in Meth A sarcomas between those transplanted into BALB/c nu/+ and into BALB/c nu/nu mice: following a single intravenous administration the effect was stronger in BALB/c nu/+ than in nu/nu mice. In contrast, tumor necrosis was almost the same in nu/+ and nu/nu mice following intratumoral administration. The present results thus indicate that TNF from mice had an antitumor activity against not only murine tumors but also human tumors. In addition to direct cytotoxicity against tumor cells, TNF induced a host-mediated factor which contributed to the antitumor effects.     

Polyethylene-glycol-modified interleukin-2 is superior to interleukin-2 in locoregional immunotherapy of established guinea-pig tumors.

Polyethylene glycol-modified recombinant human interleukin-2 (PEG-IL-2) represents a cytokine with prolonged circulatory half-life and increased antitumor activity as compared to recombinant interleukin-2 (rIL-2) after systemic administration. We studied whether PEG-IL-2 would also be advantageous in locoregional immunotherapy using a syngeneic tumor model. Intradermal inoculation of line-10 tumor cells into the flanks of strain-2 guinea-pigs results in a fast-growing tumor and regional lymph-node metastases. Treatment schedules were started on day 7 after inoculation in animals with established tumors. First, groups of 5-6 animals were treated with repeated intratumoral and perilymphatic rIL-2 or PEG-IL-2 injections. PEG-IL-2 caused significant growth inhibition of both the primary tumor and the regional lymph-node metastases at lower doses and with less frequent administration than rIL-2. The best schedule for PEG-IL-2 was 3 injections a week for 5 weeks, resulting in cure of 4/17 and 5/5 (p less than 0.01) animals at the 2 most efficient dose levels tested. Subsequent experiments indicated that the intratumoral and not the perilymphatic injection route was essential for the obtained antitumor effect. Furthermore, 12/12 animals cured after PEG-IL-2 treatment rejected a rechallenge with line-10 tumor cells, whereas no cures were seen after rIL-2 injections. PEG-IL-2 therefore appears to be a valuable substance for intratumoral immunotherapy.     

Antitumor activity of bacterial infection. II. effect of Listeria monocytogenes on growth of a guinea pig hepatoma.

Growth of a guinea pig hepatoma was suppressed when tumor cells were mixed with viable Listeria monocytogenes (LM) before intradermal (id) injection into syngeneic recipients. Heat-killed LM were less effective than viable organisms in suppressing tumor growth. A vaccine containing oil droplets and LM cell walls lacked antitumor activity. Intratumor injection of viable LM on the 7th day after id injection of tumor cells prolonged survival of guinea pigs that did not succumb to LM infection. After intratumor injection of 0.6 times 10-8-1.0 times 10-8 LM, 5 of 22 guinea pigs died from acute infection (23 percent). In the 17 survivors, 3 tumors regressed completely (18 percent). Animals surviving injections of LM and tumor cells were immune to a second challenge with tumor cells. Immunization ofguinea pigs with an intravenous injection of LM decreased the mortality from intratumor injection of LM, but the intratumor injection of LM failed to cure a significant fraction of LM-immune animals bearing 7-day hepatoma transplants. BCG was more effective than LM in producing tumor regression. Synergism between LM and BCG was not observed, and simultaneous intratumor injection of BCG and LM was no more effective than intratumor injection of BCG alone in the treatment of 12-day tumor transplants.     

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.     

DTI-015 produces cures in T9 gliosarcoma

DTI-015 (BCNU in 100% ethanol) utilizes solvent-facilitated perfusion for the intratumoral treatment of gliomas. The water-miscible organic solvent vehicle, ethanol, facilitates a rapid and thorough saturation of the'tumor with the dissolved anticancer agent, BCNU. Rats bearing established intracranial T9 gliosarcoma tumors received no treatment (group 1), a single intratumoral injection of ethanol vehicle (group 2) or DTI-015 (5 mg/kg BCNU) (group 3), or a single intratumoral injection of DTI-015 followed by systemic BCNU (group 4). Ethanol alone (n=13) had no effect on survival; MST=17 days compared to 18 days for untreated controls (n=35). DTI-015 (n=45) produced an ILS of 417% (MST=93) and 472% (MST=103) when combined with systemic BCNU (n=14). Overall, 24 of 59 rats receiving DTI-015 were judged to be cured, with 20 living a normal life span of 600 to 700 days, and 4 rats sacrificed healthy at 121, 135, 307, and 384 days post DTI-015 with no evidence of viable T9 tumor. Histology demonstrated that DTI-015 totally eradicated the T9 tumors in animals living a normal life span. The results demonstrate that a single injection of DTI-015 produces a 40% cure rate in rats bearing established intracranial T9 tumors.     

Enhanced macrophage tumoricidal activity and tumor suppression or regression caused by heat-killed Candida albicans.

The growth of line-10 hepatoma in male Sewall Wright strain 2 guinea pigs was totally suppressed when tumor cells were mixed with heat-killed Candida albicans. In a significant number of animals, injection of C. albicans into established tumors 10-12 mm in diameter caused complete, rapid tumor regression. Guinea pigs whose tumors regressed or were suppressed as a result of injection of C. albicans rejected subsequent challenges at distant sites with the line-10 hepatoma, which indicated the development of systemic immunity to the tumor. Untreated control guinea pigs had positive delayed hypersensitivity reactions to intradermally injected C. albicans, which suggested prior natural exposure of the animals to C. albicans antigens. Peritoneal macrophages from mice that had received ip injections of phosphate-buffered saline (PBS) or C. albicans were not cytocidal for mouse 3T12 tumor cells in vitro. However, macrophages from the mice given injections of C. albicans, unlike those from mice given PBS, were markedly tumoricidal in the presence of 1 ng or more endotoxin/ml in vitro. These results demonstrated that heat-killed C. albicans, when inoculated into the peritoneal cavity, increased the tumoricidal potential of peritoneal macrophages.     

Regression of established oral tumors after intralesional injection of living BCG or BCG cell walls.

Oral tumors with associated cervical lymph node metastases developed after injection of tumor cells into buccal pads of inbred guinea pigs. Intralesional injection of living BCG or BCG cell walls (CW) caused regression of established tumors, prevented the development of cervical lymph node metastases and led to the development of host resistance to the growth of subsequent tumor transplant.     

Newcastle disease virus therapy of human tumor xenografts: antitumor effects of local or systemic administration

Previously we showed that a single local injection of the avian paramyxovirus Newcastle disease virus (NDV) strain 73-T caused long-lasting, complete tumor regression of human neuroblastoma and fibrosarcoma xenografts in athymic mice. Here we report the antitumor effects of NDV administered by either the intratumoral (IT) route to treat a variety of human carcinoma xenografts or by the systemic (intraperitoneal, IP) route to treat neuroblastoma xenografts (6.5-12 mm in diameter). For IT treatments, mice were randomized into treatment groups and given a single IT injection of NDV 73-T, vehicle (phosphate buffered saline, PBS), or UV-inactivated NDV. For systemic therapy, mice (n=18) with subcutaneous IMR-32 human neuroblastoma xenografts received IP injections of NDV (5 x 10(9) PFU). Significant tumor growth inhibition (77-96%) was seen for epidermoid (KB8-5-11), colon (SW620 and HT29), large cell lung (NCIH460), breast (SKBR3), prostate (PC3), and low passage colon (MM17387) carcinoma xenografts treated IT with NDV. In all cases, tumors treated IT with PBS or replication-incompetent, UV-inactivated NDV displayed rapid tumor growth. After a single IP injection of NDV, complete regression of IMR-32 neuroblastomas was observed in 9 of 12 mice without recurrence for the 3-9 month follow-up period. Six mice with recurrent neuroblastomas after one IP injection received one to three additional IP treatments with NDV. Three of these six mice showed complete regression without recurrence. These data show that: (1) NDV administered either IT or IP is an effective antitumor therapy in this system, (2) replication competency is necessary for maximal effect, and (3) multiple NDV doses can be more effective than a single dose. These studies provide further rationale for the preclinical study of NDV as an oncolytic agent.     

Intracerebral delayed hypersensitivity reactions in glioblastoma multiforme patients.

Patients with malignant gliomas who had undergone BCG inoculation were injected intratumorally with PPD to induce an intratumoral delayed hypersensitivity reaction. Histopathological examination of the tumor before and after PPD injections revealed that chronic inflammatory responses were increased after injection in four of the five patients. In no case, however, was the response more than moderate, and in no case did the inflammatory response encompass the tumor at its peripheral margins. Intracerebral delayed hypersensitivity reactions were evoked in humans with acceptable morbidity. The mild reactions evoked did not cause detectable tumor regression.     

Widespread intratumoral virus distribution with fractionated injection enables local control of large human rhabdomyosarcoma xenografts by oncolytic herpes simplex viruses

Novel methods of local control for sarcomas are needed. We investigated the antitumor effect of two related herpes simplex virus (HSV) mutants, NV1020 and NV1066, on human rhabdomyosracoma cells and xenografts. Cell death correlated with virus replication and apoptosis in cultured cells and tumors. Complete regression was seen in all tumors <250 mm(3) following a single injection, yet only half of tumors >250 mm(3) showed a complete response. Fractionation of the virus dose into five injection sites did not increase transduction efficiency, transgene expression, or virus production, but did yield more widespread intratumoral distribution. Despite the same total dose of virus, improved control of large tumors was seen using fractionated injections as all large tumors (500-700 mm(3)) had durable, complete regression. Our data suggest that oncolytic HSVs may be useful for local control of bulky rhabdomyosarcoma tumors and that fractionated virus administration results in a more widespread virus infection and better tumor control. Therefore, strategies to maximize intratumoral virus distribution at initial delivery should be sought.     

Therapeutic vaccination against murine lymphoma by intratumoral injection of naive dendritic cells

Dendritic cells are potent antigen-presenting cells that can induce both immune responses and tolerance depending on their state of activation. Immunologic tolerance to established tumors is a major impediment for the development of effective cancer immunotherapy. Dendritic cells may be deficient in number or in function at the tumor site. To address this problem, we evaluated the ability of immature na?ve dendritic cells to induce an antitumor immune response when injected directly into a murine B-cell lymphoma. Mice with advanced transplanted syngeneic tumor were given intratumoral injections of bone marrow-derived dendritic cells. Intratumoral dendritic cell injection alone had no antitumor effect. Systemic chemotherapy alone resulted in only transient tumor regression. However, the intratumoral injection of dendritic cells after chemotherapy led to complete, long-term tumor regression in the majority of treated mice. This dendritic cell-mediated antitumor effect was systemic, resulting in simultaneous elimination of the tumor at second uninjected sites. In addition, it resulted in long-term memory with resistance to tumor rechallenge. Both CD4+ and CD8+ T cells are necessary for the antitumor effect. Furthermore, tumors that occasionally recurred in mice with initial complete tumor regression could be retreated by the same combined chemoimmunotherapy approach. These results show that immunotherapy can succeed in the setting of advanced lymphoma if dendritic cells are restored and loaded with tumor antigens in situ at a single tumor site.     

Responses of tumors induced in inbred guinea pig strain JY=1 and strain Hartley/F to BCG.

A transplantable fibrosarcoma induced in inbred JY-1 guinea pig strain by 3-methylcholanthrene (MCA) and designated J4, an allotransplantable subline of J4 (JH4) which was obtained by the transplantation of J4 into the inbred Hartley/F guinea pig strain and maintained by passages in this strain, and a syngeneic liposarcoma H10 induced in a Hartley/F guinea pig by MCA were tested for their immunotherapeutic response with BCG. The growth of J4 and H10 tumors was suppressed in most of the animals when tumor cells were mixed with BCG before being injected sc into BCG-immune or BCG-nonimmune recipients. The growth of the JH4 tumor was suppressed at the sites of injection with a mixture of tumor cells and BCG in BCG-immune recipients but not in nonimmune animals. All guinea pigs surviving the injection of a tumor cell-BCG mixture resisted a second tumor cell challenge. When subcutaneous sarcomas grew to about 8-15 mm in diameter, BCG was injected into the tumors. The growth of JH4 tumor was not influenced by the injection in either BCG-immune or BCG-nonimmune animals, while the regression of the established J4 transplants was produced in 2 of 3 nonimmune recipients. The growth of the H10 tumor was not inhibited with an intratumor injection into nonimmune guinea pigs, while the H10 tumor regressed in BCG-immune animals for 4-5 weeks after intratumor injection and thereafter grew progressively. Skin reactions in animals that received repeated intradermal injections of the tumor cells and BCG were tested with 10(6) viable tumor cells as eliciting antigens. Typical delayed-type hypersensitivity reactions that were specific to the homologous antigens were observed. The possible reasons for the different responses to BCG among the guinea pig tumors, including line-10 hepatocarcinoma in strain-2 guinea pigs, were discussed.