Antitumour responses induced by short-term pretreatment with tumour cells.

The injection (s.c. or i.p.) of 10(6) live or lethally irradiated methylcholanthrene-induced fibrosarcoma cells into CBA/Ca mice one or 2 days before i.v. challenge with the same tumour inhibited the formation of artificial lung tumour metastases. In addition, it also frequently enhanced the cytostatic effect of peritoneal exudate cells on monolayers of the same tumour. The effects on lung tumour metastasis were not noted if X-irradiated tumour was injected i.v., or if s.c. administration was delayed until one day after i.v. challenge. Similar effects on tumour growth were also observed in C3Hf/Bu mice and (CBA/Ca x A/HeJ) F1 hybrids which were pretreated (s.c.) with tumour shortly before i.v. challenge with the same tumour. Further studies in CBA/Ca mice suggested that the protective effect was tumour-specific, for the growth of i.v. injected tumour was not significantly inhibited by pretreatement with a number of other MC-induced or spontaneous tumours from the same and different strains.     

Active specific immunotherapy of mouse methylcholanthrene induced tumours with Corynebacterium parvum and irradiated tumour cells.

The relative efficiency of active nonspecific or specific immunotherapy of developing methylcholanthrene induced fibrosarcomata with C. parvum was compared. For nonspecific immunotherapy, mice were challenged with tumour cells s.c. or i.v., and 2 days later injected i.v. with dilutions of C. parvum. The only significant effect was a retardation of s.c. tumour growth by the highest concentration of C. parvum (350 mug). However, active specific immunotherapy, using mixtures of C. parvum and irradiated or living tumour cells in the footpads, suppressed tumour growth when given at 2 or 6, but not 10, days after tumour challenge. Successful therapy required: sufficient tumour cells (greater than or equal to 5 X 10(4)); an optimal dose of C. parvum (5-120 mug, increasing with the number of tumour cells); an intact T cell system; the same tumour cells for challenge and treatment. The specificity was confirmed in a protection system in which treatment was given 7 days before tumour challenge. No protective immunity could be achieved with mixtures of C. parvum and foetal cells. Thus in this system C. parvum potentiates protective immunity only to the tumour unique TSTA.     

Effects of C. parvum on growth and induction of intracerebral tumours in mice.

An investigation was made into the effect of Corynebacterium parvum therapy on cerebral tumours in mice. I.v. C. parvum caused a slight but significant increase in the survival of BALB/c mice injected intracerebrally (i.c.) with not more than 50 Meth A cells. C. parvum was most effective if given on the same day or 5 days after tumour. If this interval was increased there was no effect. Multiple i.v. injections were no more effective than a single dose. I.v. C. parvum had no influence on the survival of C57BL mice injected i.c. with Lewis tumour cells, and had little effect on the induction of i.c. or s.c. tumours by methylcholanthrene. It was concluded that C. parvum therapy was of little use in the treatment of cerebral tumour in mice. The clinical implications of these findings are discussed.     

Effect of toxic thioureas on resistance of rats to growth in the lungs of intravenously and intratracheally seeded tumour cells.

Clonogenic growth (colony-forming efficiency, CFE) of i.v. injected allogeneic W256 tumour cells in the lungs was markedly enhanced by treatment of rats with alpha-naphthyl thiourea (ANTU) injected i.p. from 2 h before to 2 h after the tumour cells. ANTU specifically increases pulmonary vascular permeability in adult rats and causes acute pulmonary oedema and pleural effusion. Inhibition of drug toxicity to the lungs by tachyphylaxis, specific antimetabolites or iodides did not abolish the effect of ANTU on CFE. CFE was not increased when cells were seeded by i.v. injection the lungs affected by advanced pulmonary oedema at 6 to 24 h after treatment with drug. ANTU did not enhance growth of intratracheally injected cells. Although ANTU has no cytotoxic or immunosuppressive action, treatment of tumour-immunized rats with ANTU caused apparent "breakdown" of tumour immunity in 50% of rats, by causing growth of tumour colonies in the lungs. Possible mechanisms for the ANTU-induced decrease in innate resistance to growth of tumour in the lungs are discussed.     

Temporary inhibition of Moloney-murine sarcoma virus (M-MSV) induced-tumours by adoptive transfer of ricin-treated T-lymphocytes

The potential use of tumour-specific T-lymphocytes loaded with ricin in cell targeting experiments was investigated. Moloney-murine sarcoma virus (M-MSV)-specific T-lymphocytes, obtained in mass mixed leucocyte-tumour cell culture (MLTC) and a M-MSV-specific cytotoxic T-lymphocyte (CTL) clone, were incubated with 125I-labelled ricin in order to evaluate toxin uptake and release. The internalized ricin (4.5 X 10(-17) mol and 6.5 X 10(-17) mol per 10(2) MLTC and CTL clone cells, respectively) was released rapidly during the first 30 min following treatment, and at a constant but slower rate over the next few hours. The cytotoxic activity of ricin-treated cells evaluated against antigen-related target cells, in a short term incubation 51Cr release assay, was unaffected during the first 30 min after treatment but decreased with time over the next few hours. However, the growth of antigen related as well as of unrelated tumour cells was strongly inhibited by the addition of ricin-treated cells to the culture system, thus indicating that released ricin is toxic for untreated target cells. The in vivo localization pattern of ricin-treated radiolabelled MLTC cells was found to be comparable with that of untreated cells 1 h after i.v. injection into syngeneic sublethally irradiated mice. After 6 h, however, more radiolabel was recovered from the liver of mice receiving ricin-treated MLTC cells. Ricin-treated M-MSV-specific T-lymphocytes were injected i.v. into tumour bearing sublethally irradiated mice. A temporary tumour growth inhibition (up to 6 days) was achieved following transfer of low doses of ricin-treated MLTC or CTL clone cells (1 X 10(6) and 0.5 X 10(6), respectively). In contrast, in M-MSV injected control mice, receiving only free toxin (from 5 to 20 ng) or untreated tumour-specific effector cell tumours grew progressively. The therapeutic effect was apparently specific since the injection of ricin-treated alloreactive T-lymphocytes did not influence tumour growth. These results suggest that M-MSV-specific T-lymphocytes loaded with ricin can deliver toxin to the target tumour mass and have a transient therapeutic effect.     

The presence of a tumour in F1 mice partially inhibits the GvH reaction following injection of parental spleen cells.

(A x CBA(T6)F1 mice bearing F1 mammary carcinomas for 9 days were injected i.v. with A strain spleen cells. The A spleen cells were from either non-immune donors or mice which had received an i.p. injection of F1 tumours cells 9 days previously, and were thus immune to the CBA component of the tumour. Fourteen days after receiving parental spleen cells, the F1-tumour-bearing mice were killed and their spleen ratios and indices were determined as an index of the severity of the graft-versus-host reaction (GvHR) induced. The spleen indices were compared with those in non-tumour-bearing F1 mice, receiving aliquots of the same parental cell suspensions. At the higher doses of A spleen cells, the presence of an F1 tumour reduced the GvHR. At the same time, in 3/5 experiments, the weight of the F1 tumour in mice injected with immune A spleen cells was less than that in F1 mice receiving the same number of tumour cells but no spleen cells. A reduction in GvHR and a decrease in tumour weight was not seen when F1 mice carrying an A strain tumour were injected with A strain spleen cells immune to an F1 tumour. Adding 23-day F1 tumour-bearing F1 spleen cells to A spleen cells did not reduce the GvHR induced in further non-tumour-bearing F1 recipients by the parental cells. This was evidence against the presence of suppressor cells in the tumour-bearing F1 spleen. When 51Cr-labelled A strain spleen cells were injected into F1 mice, some of which had a tumour and therefore an enlarged spleen, there was an inverse relation between the size of the spleen and the number of parental cells therein, per g spleen 4 h after injection. It is thus suggested that the reduction in GvHR in F1-tumour-bearing F1 mice, after injection of parental spleen cells, is due first to a reduction in the concentration of donor cells in the recipient spleen (i.e. the same number of donor cells in a larger spleen) and second to pre-occupation of the donor cells in reacting to the tumour.     

Effect of age and sex on lung-colony-forming efficiency of injected mouse tumour cells.

The i.v. injection of a specified number of cells of either an Ehrlich ascites tumour (ELD) or spontaneous mouse mammary adenocarcinomas (MA) into C3H mice yielded a number of lung colonies which varied significantly with the age or sex of recipient mice. The yield was higher in mice of 71 weeks than in those of 15 weeks, except for MA cells injected into females, when the yield was higher in the younger mice. Sex did not influence very significantly the yield of colonies from ELD cells; in the case of MA cells the direction of sex differences depended on age. A difference in the effect of pre-immunization with age was not observed.     

Low density lipoprotein for delivery of a water-insoluble alkylating agent to malignant cells. In vitro and in vivo studies of a drug-lipoprotein complex

Previous studies have shown that human leukaemic cells and certain tumour tissues have a higher receptor-mediated uptake of low density lipoprotein (LDL) than the corresponding normal cells or tissues. LDL has therefore been proposed as a carrier for anti-cancer agents. In the current study, a water-insoluble mitoclomine derivative (WB 4291) was incorporated into LDL. The WB 4291-LDL complex contained about 1,500 drug molecules per LDL particle and showed receptor-mediated toxicity in vitro as judged from the difference in growth inhibitory effect on normal and mutant (LDL-receptor-negative) cultured Chinese hamster ovary cells. However, cellular drug uptake did not exclusively occur by the receptor pathway since mutant cells were also affected to some extent. The LDL part of the complex had the same plasma clearance and organ distribution as native LDL after i.v. injection in mice and rabbits. Therapeutic effects were observed when Balb-C mice with experimental leukaemia were treated with the complex. After i.p. administration to mice with i.p. leukaemia median survival time was prolonged 2.5-fold and 40% became long time survivors. The effect was weaker (42% increase in life span) after i.v. injections of the complex to mice with i.v. leukaemia.     

Growth of a transplantable lymphoma and its modification in mice infected with the inducing virus.

The growth of a transplantable lymphoma was examined in normal mice and in mice previously infected with the lymphoma-inducing virus (ULV). Normal BALB/c mice respond to a footpad injection of X-irradiated lymphoma cells (ULMC) with popliteal lymph node (PLN) enlargement; mice previously infected with ULV do not. 106 viable ULMC injected into the footpads of ULV-infected mice grew progressively, and the animals died with disseminating malignant lymphoma. In contrast, this dose of cells injected into normal animals evoked strong host responses in the foot and draining lymph node, and no progressive growth of the lymphoma occurred. This increased susceptibility of the ULV-infected animals was also observed when ULMC were injected s.c. into the back or i.m. into the calf muscle, but not after s.c. injection of an unrelated 3-methylcholanthrene-induced sarcoma. Resistance to tumour growth after i.v. injection of ULMC is clearly ineffective, since 10 cells can grow and kill the animal, and in this case no increased susceptibility of ULV-infected animals was observed.     

Foscan-mediated photodynamic therapy for a peritoneal-cancer model: Drug distribution and efficacy studies

Distribution of the photosensitizer Foscan® (meta-tetrahydroxyphenylchlorin, mTHPC), after i.v. or i.p. injection, was investigated in Wag/Rij rats bearing i.p. tumours. These results were compared with the efficacy of mTHPC-mediated photodynamic therapy for illumination intervals of 4 hr to 3 days. For the distribution experiments a single tumour (CC531 colon carcinoma) was implanted intra-abdominally in a fat pad, or a cell suspension (1 × 10⁶ CC531 cells) was injected into the peritoneal cavity, which results in a dissemination of tumour nodules on the peritoneum. ¹⁴C-mTHPC was not selectively taken up in the single-tumour model after i.v. or i.p. injection, but higher concentrations were achieved for i.p. administration. For this tumour model the concentration ratios between tumour and normal tissue never exceeded a value of 3. In the disseminated-tumour model, an uptake of up to 40% of the injected dose was found per gram tumour at 4 hr after an i.p. injection and this resulted in very high (>14) concentration ratios of tumour to normal tissues. Low uptake was found after the i.v. injection route (1% of the injected dose per gram tumour) with lower tumour/normal tissue ratios (<8). The efficacy of i.p. photodynamic therapy (IPPDT) was evaluated using the single-tumour model only. The lower abdomen was illuminated at 4 hr to 3 days after mTHPC, and tumour size was repeatedly measured via a small laparoscopy. Significant delay in tumour regrowth was achieved for 6 J · cm⁻² at 1 day after i.v., or at 4 hr after i.p. mTHPC (p values 0.019 and 0.045 respectively). Response to PDT, of tumours implanted in the fat pad, was not greater for i.p. administration of the photosensitizer and there was a poor correlation between times of maximum drug uptake in tumours and optimal illumination times for PDT efficacy. Int. J. Cancer 73:230–235, 1997. © 1997 Wiley-Liss, Inc.     

Possible role of macrophage-like suppressor cells in the anti-tumour activity of BCG

The i.v. injection of high doses (3 mg) of BCG into C3H mice bearing a transplantable 3-methylcholanthrene-induced fibrosarcoma caused the regression of a significant proportion. This effect was most evident when the BCG was injected on the day of the graft, or 7 days later. The injection of this agent either 14 days before the graft, or in low doses (0.1 or 0.5 mg), or directly into the tumour (i.t.) only prolonged the survival of the animals. Spleen cells from systemic high-dose BCG-treated mice were found to exert a strong nonspecific cytostatic effect in vitro that was not an artefact of the test conditions, and was not expressed by cells from low-dose animals. The cytostatic effect was shown to be caused by cells with the characteristics of macrophages, i.e. they were strongly adherent, unaffected by treatment with anti-Thy 1.2 + C', radioresistant but heat-sensitive, and were detected in BCG-treated "B" mice. The spleens of high-dose BCG-treated mice also contained suppressor cells that were capable of inhibiting the in vitro reactivity of normal T cells to PHA. Like the cytostatic effect, this suppressor activity was not detected in low-dose mice, and the cells responsible had the properties of macrophages; the effect was lost after the removal of adherent cells by sequential exposure to plastic and colloidal iron, but was conserved after treatment with anti-Thy 1.2 + C'. T-cell-deprived animals, such as "B" or nude mice, also developed suppressor-cell activity when treated with systemic high-dose BCG. Close parallels became evident between the in vivo anti-tumour activity of BCG, the in vitro cytostatic effect, and the suppressor-cell activity. We here discuss the possible role of suppressor cells in the mechanism of action of this agent.     

Host treatments affecting artificial pulmonary metastases: interpretation of loss of radioactively labelled cells from lungs

The effect has been examined of various host treatments (C. parvum injection, immunization, thoracic irradiation, cyclophosphamide injection, and anticoagulation) on both lung colony formation and clearance of radioactive cells from the lungs after i.v. injection of tumour cells. Two tumour-host models have been used: the non-immunogenic KHT tumour in C3H/Km mice, and the immunogenic EMT6 tumour in BALB/c/Ka mice. Even for the at most weakly immunogenic KHT tumour, the number of artificial pulmonary metastases could be modified by a factor of up to 10(4) by different host treatments before i.v. inoculation of tumour cells. For all pretreatments except immunization, the shape of the curve of loss of radioactivity from the lungs vs time was biphasic, with an initial steep portion representing intravascular death of the tumor cells, followed 1--2 days after tumour-cell injection by a shallow exponential curve. It was concluded that the shallow slope represented spontaneous death of tumour cells in the perivascular tissues. Essentially all the injected tumour cells lodged initially in the lungs, and this was unaffected by the different host treatments. Furthermore, except for specific immunization, cell death in the perivascular tissues was also unaffected by host treatment. However, the survival of the tumour cells during the 24 h after injection (before they became extravascular) was extremely dependent on the particular host pretreatment. It would appear from these studies that host treatments such as C. parvum injection or anticoagulation can markedly affect the number of blood-borne pulmonary metastases, but they will only be effective if given before the tumor cells arrive in the lung vasculature.     

Treatment of minimal residual disease after surgery or chemotherapy in mice carrying HPV16-associated tumours: Cytokine and gene therapy with IL-2 and GM-CSF

Moderately immunogenic HPV16-associated tumours TC-1 (MHC class I+, HPV16 E6/E7+, G12V Ha-ras+) and MK16/1/IIIABC (MK16, MHC class I-, HPV16 E6/E7+, G12V Ha-ras+), both of the H-2b haplotype and transplanted in syngeneic mice, were used to examine the effects of local IL-2 and GM-CSF cytokine or gene therapy in the treatment of minimal residual tumour disease. The mice carrying MHC class I+ TC-1 tumour residua after surgery were injected into the site of the surgery either with irradiated, IL-2 gene-modified MK16 tumour cells, or with recombinant IL-2. It has been found that both, the recombinant IL-2 and the IL-2 gene-modified tumour vaccine substantially reduced the percentage of tumour recurrences in the operated mice. Similarly, when the mice carrying TC-1 tumour residua after surgery were injected with recombinant GM-CSF, the recombinant GM-CSF inhibited growth of the tumour residua in the operated mice. Gene therapy with irradiated, GM-CSF secreting MK16 cells did not produce any tumour-inhibitory effect. In further experiments, mice bearing s.c. TC-1 tumours were injected i.p. with ifosfamide derivative CBM-4A and 8 days later, peritumourally, either with IL-2 gene-modified and IL-2-producing MK16 cells, or with recombinant IL-2. It has been found that both, the recombinant IL-2 and the IL-2 gene therapy substantially reduced the percentage of tumour-bearing mice. When the mice bearing s.c. TC-1 tumours were injected i.p. with ifosfamide derivative CBM-4A and then, peritumourally, either with irradiated, GM-CSF gene-modified and GM-CSF-producing MK16 cells, or with recombinant GM-CSF, it was found that both, the recombinant GM-CSF and GM-CSF gene therapy inhibited growth of tumour residua. Comparative experiments were performed with the MHC class I-, metastasizing tumour MK16. It has been found that both, recombinant IL-2 and GM-CSF, can inhibit growth of the tumour residua after surgery or chemotherapy. The lung metastases in mice with surgical minimal residual tumour disease or in mice with tumour residua after chemotherapy were inhibited by IL-2 but not by GM-CSF. The MK16 tumour vaccine producing IL-2 inhibited growth of tumour residua after chemotherapy, but not the tumour residua after surgery. The GM-CSF-producing vaccine was without significant effect in both, surgically- and chemotherapeutically-induced minimal residual MK16 tumour disease. In conclusion, the MHC class I+ and MHC class I-, HPV16-associated tumours were found to be sensitive to IL-2 and GM-CSF therapy after surgery or after cytoreductive chemotherapy. It is yet to be addressed if this is more general case with HPV16-associated experimental tumours. If so, it would be of interest to further investigate whether such adjuvant therapy can also help to eradicate the residua after surgery and chemotherapy in patients carrying HPV16-associated neoplasms.     

Enhancement of experimental metastasis by pretreatment of tumour cells with hydroxyurea

The effect of treatment with hydroxyurea (HU), on subsequent experimental metastasis formation by cells of 3 murine melanoma lines has been examined. In vitro treatment of B16-F1, B16-F10 and K-1735-clone 19 cells with 0.1 mM or 0.3 mM HU, followed by a period of recovery in drug-free medium, resulted in a significant increase in the number of lung nodules formed in syngeneic mice following i.v. injection of the cells. The maximum effect obtained was observed when cells were injected 6 hr after removal of HU and it occurred in spite of the cytotoxicity of the doses used. Six hours after release from treatment with 0.3 mM HU cells were synchronized in the G2 phase of the cell cycle but the consequent increase in cell volume was not responsible for increased metastasis through enhancement of tumour-cell arrest, since lodgement of 51Cr-labelled, HU-treated cells was no greater than that of control cells. The results obtained suggest that the chemotherapeutic agent HU may facilitate tumour progression via a direct action on neoplastic cells.     

Distant metastasis facilitated by BCG: spread of tumour cells injected in the BCG-primed site.

Tumour metastasis in BCG-pretreated mice was studied using a methylcholanthrene-induced fibrosarcoma in C3H/He mice. When tumour cells were injected into the BCG-primed site, distant metastasis occurred in the lungs and the popliteal lymph node, through this tumour did not metastasize in normal mice. Such metastases were increased in proportion to the number of tumour cells injected into the BCG-primed site, and developed soon after tumour challenge. Concomitant immunity developed well in the mice bearing such metastases, but did not inhibit metastatic growth. Experiments using 125I-labelled SRBC or tumour cells revealed that such cells egressed rapidly from the BCG-primed site. When the tumour was inoculated into the contralateral foot to the BCG-primed site, the incidence and the number of metastases was reduced. Furthermore, BCG infection induced an increase of platelet count. I.v. injection of this tumour induced marked thrombocytopenia in normal mice. Administration of pentoxifylline, a methylxanthine derivative before tumour challenge reduced such metastases. These findings suggest that the changes in peripheral blood, such as increased platelet count and increased release of tumour cells from the injection site, facilitated distant metastasis in BCG-pretreated mice.     

Enhancement by cytotoxic agents of artificial pulmonary metastasis.

The formation of lung colonies by i.v. injected Lewis lung-tumour cells in syngeneic recipients was greatly enhanced by prior treatment of the mice with cyclophosphamide. The lung-cloning efficiency was linearly related to cyclophosphamide dose and the optimum time of treatment was 2-4 days before the injection of tumour cells. The resulting lung colonies had a similar size distribution to colonies in untreated recipients. Bleomycin, local thoraric irradiation and whole-body irradiation were much less effective in enhancing the lung-cloning efficiency. Cyclophosphamide also enhanced the take probability of i.m. implanted tumour cells.     

TZT-1027 elucidates antitumor activity through direct cytotoxicity and selective blockade of blood supply

BACKGROUND: TZT-1027 is a newly developed antitumor agent derived from dolastatin 10. MATERIALS AND METHODS: The in vitro activity of TZT-1027 on MCF-7 and R-27 cells was evaluated by MTT assay. TZT-1027 1 mg/kg/week was administered i.v. for 4 weeks into nude mice bearing MCF-7 and R-27. Subsequently, primary cultured cells from xenografts were also used for CD-DST. Two mg of TZT-1027 or 40 mg docetaxel per kg were injected i.v. into nude mice bearing R-27. 0.2% Evans blue was injected to assess the blood flow. RESULTS: TZT-1027 suppressed the in vitro growth of MCF-7 cells, while R-27 cells were resistant to TZT-1027, although its in vivo antitumor activity was remarkable. TZT-1027 blockaded R-27 tumor blood flow immediately after injection; blood flow was not affected by docetaxel. CONCLUSION: TZT-1027 exerts its antitumor activity through direct cytotoxicity against MCF-7 cells and through selective blockade of tumor blood flow against R-27 cells.     

Influence of C. parvum on the effectiveness of passive serotherapy in the control of the EL4 lymphoma in C57BL/6 mice.

Administration of C. parvum alone did not improve the survival of C57BL/6 mice injected with the EL4 lymphoma. The anti-tumour effect of anti-EL4 Ig was however increased by C. parvum treatment, and the combination therapy of anti-EL4 Ig and cytotoxic drugs was even more improved. However, C. parvum only had this effect when given by the same i.p. route as the tumour cells, and the effect was greater when C. parvum was injected 5 days before than 1 day after tumour cells.     

Immunisation with 'naïve' syngeneic dendritic cells protects mice from tumour challenge

Dendritic cells (DCs) ‘pulsed'' with an appropriate antigen may elicit an antitumour immune response in mouse models. However, while attempting to develop a DC immunotherapy protocol for the treatment of breast cancer based on the tumour-associated MUC1 glycoforms, we found that unpulsed DCs can affect tumour growth. Protection from RMA-MUC1 tumour challenge was achieved in C57Bl/6 MUC1 transgenic mice by immunising with syngeneic DCs pulsed with a MUC1 peptide. However, unpulsed DCs gave a similar level of protection, making it impossible to evaluate the effect of immunisation of mice with DCs pulsed with the specific peptide. Balb/C mice could also be protected from tumour challenge by immunisation with unpulsed DCs prior to challenge with murine mammary tumour cells (410.4) or these cells transfected with MUC1 (E3). Protection was achieved with as few as three injections of 50 000 naïve DCs per mouse per week, was not dependent on injection route, and was not specific to cell lines expressing human MUC1. However, the use of Rag2-knockout mice demonstrated that the adaptive immune response was required for tumour rejection. Injection of unpulsed DCs into mice bearing the E3 tumour slowed tumour growth. In vitro, production of IFN-γ and IL-4 was increased in splenic cells isolated from mice immunised with DCs. Depleting CD4 T cells in vitro partially decreased cytokine production by splenocytes, but CD8 depletion had no effect. This paper shows that naïve syngeneic DCs may induce an antitumour immune response and has implications for DC immunotherapy preclinical and clinical trials.     

Radiolabelling of Corynebacterium parvum and its distribution in mice.

Corynebacterium parvum was labelled by growing live bacteria in the presence of [3H]thymidine. The bacteria were killed by formalin, washed thoroughly and resuspended at a concentration of 7 mg dry weight/ml. An activity of 1-6 X 10(5) ct/min/0-1 ml was obtained. The biological properties (inhibition of tumour growth and hepatosplenomegaly) of the labelled C. parvum were compared with those of commercially available vaccine, and were found to be similar. Labelled C. parvum was injected i.v., i.p., or s.c. into normal C57BL mice and the localization of activity determined at 4 h and 1,3,7 and 14 days after injection. After i.v. or i.p. injection, highest counts were recorded in the liver. Moderate activity was found in the spleen, lungs and small gut. After s.c. injection, the majority of radioactive label was detected at the site of injection and little found in other tissues. The distribution of injected C. parvum was also studied in mice bearing Lewis tumour, and was found to be similar to that in normal mice. Moderate amounts of labelled C. parvum were recovered from tumour. There appeared to be no relationship between the antitumour effect of C. parvum given by a particular route of injection and the concentration of C. parvum recovered from the tumour.