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Although the hematoporphyrin derivative (Hpd) is one of the most studied photosensitisers for photodynamic therapy (PDT), it is far from ideal. Therefore, many laboratories have been investigating a new group of sensitisers, the phthalocyanines. Particularly, in our laboratory we decided to study the aluminum disulfonated phthalocyanines (AlS2PC). They are chemically stable, readily soluble in water and have a strong absorption in the red part of the spectrum at 675 nm. Mice bearing the MS-2 fibrosarcoma treated with 5 mg/kg of AlS2PC survived indefinitely also using a low laser power of 100 mW/cm2 X 10' of exposure time, in contrast to experiments carried out with Hpd where the optical therapeutic laser power was 400 mW/cm2 X 10' and the dose of Hpd was 25 mg/kg. Furthermore, treatment of mice bearing the highly metastatic tumor, B16 melanoma, with 5 mg/kg of AlS2PC and laser light (100 mW/cm2 X 10'), significantly prolonged the survival time in respect to mice treated with 25 mg/kg of Hpd and laser light (400 mW/cm2 X 10').

Time-gated fluorescence spectroscopy was performed on the tumour-localizing fraction (TLF) of haematoporphyrin derivative (HPD) incorporated into cells. Three different cell lines were incubated with 20 and 5 micrograms ml-1 of TLF for various time periods; they were then washed and resuspended in buffer. Fluorescence decay measurements and time-integrated and time-gated spectra were then obtained from the cell suspensions. Similar experiments were repeated using HPD containing 60% of the active material. The experimental results show a modification of the emission spectra for both drugs depending on the incubation time; this modification is more significant for the TLF. In particular, the emission peak observed in aqueous solution at 615 nm is shifted to 630 nm as a consequence of incorporation into cells, and the gated spectra indicate that the fluorescence emission is mainly related to monomers and unfolded polymeric chains. The ratio between the intensities of the two peaks depends on the relative amount of the TLF; the peak at 615 nm is more pronounced for HPD. The results obtained seem to indicate that both the composition of the drug and the metabolic properties of the biological environment strongly influence the uptake process and the fluorescence behaviour of the incorporated sensitizer.

A fluorescence imaging system was used to monitor the emission of disulfonated aluminum phthalocyanine (AlS2Pc) during the photodynamic therapy (PDT) of murine tumors. Cells of the MS-2 fibrosarcoma were injected in mice in two compartments in order to cause the development of tumors in different host tissues. Two drug doses and two uptake times were considered. Moreover, the fluorescence of the AlS 2Pc was excited using two wavelengths on the opposite sides of the absorption peak to detect a possible change in the absorption spectrum of the sensitizer induced by the PDT. In the tumors, the treatment induces a variation of the fluorescence intensity: in some mice a mild photobleaching takes place, in others a fluorescence enhancement occurs. Which effect predominates depends on the experimental conditions, even though a large spread of data was found amongst mice of the same group. In all mice, independently of the drug dose, uptake time or tumor compartment, a marked increase in the fluorescence signal takes place at the borders of the irradiated area. To quantify this effect we evaluated the ratio between the fluorescence intensities in the peritumoral area and in the tumor itself. This ratio increases monotonically during the PDT, showing a different behavior with the two excitation wavelengths. This indicates that the AlS 2Pc absorption spectrum shifts toward shorter wavelengths as a result of the irradiation.

In vivo treatment of mouse leukemia L1210 with DTIC can induce new antigens on tumor cells that are not detectable on parental cells and that are transmissible as a genetic character. Moreover, L1210/DTIC is rejected by syngeneic hosts. The aim of this study was to investigate whether DTIC selects pre-existing immunogenic clones rather than inducing ex novo new antigenic determinants and to verify the number of induced antigens. L1210 leukemia was cloned in vitro and 4 clones were treated in vivo with DTIC. All the treated clones displayed antigenic properties since they were rejected by syngeneic hosts. Cytotoxic T lymphocytes (CTL) activated against one DTIC clone could recognize and lyse the relevant target. One of these DTIC-modified clones (L4/DTIC) was recloned and the subclones were tested in vivo and in vitro. Two out of six subclones were rejected by syngeneic hosts. CTL specific against these two clones were able to recognize and lyse all the other clones to different degrees. The degree of suscptibility to lysis did not correlate with the capability to evoke an immune response in vivo. Based on these findings we conclude that DTIC does not select pre-existing clones but modifies the tumor cells antigenically, and that the antigenicity induced by DTIC in a cloned tumor line is due to the presence of common antigens shared to different degrees with treated cells.

Hematoporphyrin derivative (Hpd) is a fluorescent dye that is preferentially incorporated by tissues with a high mitotic index, such as tumor cells and blast cells. A cytotoxic effect is produced following light activation. Previous studies have shown a long lasting reversible inhibition of DNA synthesis in Hpd-treated cells that failed to stimulate allogeneic lymphocytes in either primary or in secondary MLR. In this study we report Hpd inhibitory effects on some immunological assays in vitro. Treatment with Hpd of cytotoxic effector cells resulted in inhibition of their lytic activity likely dependent on the loss of binding to target cells. In the same way Hpd treatment inactivated the lytic activity of NK cells. In contrast Hpd-treatment of target cells did not modify the above immunological reactions. Moreover Con A agglutinability, antibody dependent capping as well as E-rosettes were inhibited following an Hpd treatment of relevant cells. Since normal susceptibility to humoral and cell mediated lysis was exhibited by Hpd-treated cells it is unlikely that cell surface molecules were damaged. An inhibitory effect exerted by an Hpd treatment on cell surface movements might explain these findings.

Summary A tumour line inoculated in mice showed high affinity binding for lipoproteins in vitro. Studies in vivo demonstrated that the assimilation of human low density lipoprotein (LDL) by the tumour was very high. Both receptor and non-receptor mediated catabolism of the lipoprotein by the tumour increased as compared to other tissues known to be sites of lipoprotein catabolism (liver, spleen etc.). These findings suggest that lipoproteins may be useful markers for tumours as well as carriers for cytotoxic drugs to target tissues in vivo.

SUMMARY The experiments reported here concern the charcterization by techniques of in vitro cell-mediated immunity of the antigens induced by 5-(3,3'dimethyl-1-triazine)-imidazole-4-carboxamide (DTIC) on L1210, a chemically-induced lymphoma of DBA/2 mice (H-2d). This series of experiments with the DTIC-treated L1210 tumour show the presence of an H-2D'-like antigen which resembles the Dk gene product/s of the H-2k haplotype.

Polychlorinated biphenyls (PCBs) are environmental contaminants, whose toxicity is related to the degree and the position of chlorine substitutes. Fenclor 42 is a mixture of trichlorobiphenyls employed for industrial use in Italy. In order to evaluate its effects on the immune system, CD2F1 mice were treated with Fenclor 42 at different doses and schedules. Spleen weights, total number of splenocytes and relative spleen weight decreased significatively following a single or a prolonged exposure to different doses of Fenclor 42. An analysis of the functional activity of splenocytes showed that the proliferative response to Con A and PWM mitogens was also inhibited by Fenclor 42. Splenic parameters returned to normal values within 5 days after a single injection and between 8–15 days after a subchronic administration of the compound. The capability of splenocytes to proliferate, following mitogen stimulation, was restored between day 5 and day 8 according to the different schedules of treatment. By comparison, Fenclor 42 never affected NK activity. These data clearly indicate damage at the splenic level by Fenclor 42. Studies are in progress to elucidate the mechanisms involved in the immunotoxic response.

Antineoplastic compounds can induce on tumour cells new antigens that undetectable on parental cells and which are transmissible as a genetic character. In this study mouse leukaemia L1210 was cloned in vitro by limiting dilution and one cloned line was recloned in vivo. Four subcloned tumour cell lines (A,D,R,S) were xenogenized in vivo by DTIC treatment (A/DTIC, D/DTIC, R/DTIC, S/DTIC) following a schedule previously described. Up to 10(7) cells of these xenogenized subclones, injected i.p., were rejected by syngeneic hosts, although they grew in immunosuppressed hosts. The DTIC treated subclones were lysed by in vivo-primed, in vitro-restimulated (with the relevant subclone) lymphocytes. The cytotoxic lymphocyte activity was not strictly specific since parental, DTIC-untreated cells were also lysed, although less efficiently. CTL directed against the D/DTIC subclone were cloned by limiting dilution. Ninety-four CTL clones were assayed against L1210 subcloned cells, DTIC-treated and untreated, and against different murine tumours (syngeneic or allogenic). Three specific antigens could be identified in the 51Cr release assay. The DTIC subclones expressed one antigen that was specifically recognized by a set of CTL clones. A number of CTL clones were able to lyse the L1210 subcloned cell exclusively, targetting a tumour-associated antigen that did not appear to be modified in the DTIC-treated subclones. A third antigen was demonstrated in the parental and DTIC treated D subclone. On the basis of these results it was postulated that there was at least one common DTIC-inducible antigen specific and reproducible within an identical cell population. Moreover, DTIC treatment did not modify histocompatibility antigens or TAA pre-existing in L1210 cells. The findings discussed here provide new information about permanent xenogenization of tumour cells, which might be exploited for experimental chemo-immunotherapy of cancer.

The aim of this study is to evaluate the effects of Fenclor 42 (a mixture of trichlorobiphenyls) on the immune system. A prolonged administration of this compound to CD2F1 mice resulted in a reduction of relative spleen and thymus weight according to the dose. Furthermore, spleen weights, total number of splenocytes and relative spleen weights decreased significantly also following a single treatment with 0.5 g/kg or 1 g/kg of Fenclor 42. An analysis of the functional activity of splenocytes pointed out that proliferative response to mitogens was also inhibited. Splenic parameters returned to normal values within 5 days after a single treatment and between 8 and 15 days after a subchronic administration. The functional activity of splenocytes was restored between day +5 and day +8 according to the different schedules of treatment. On the contrary, natural killer cell (NK) activity was never affected by Fenclor 42. Studies are in progress to elucidate the intimate mechanism of the toxicity of Fenclor 42 on immunocompetent cells.

Treatment of murine tumors with the anti-tumor agent 5-(3,3 dimethyl-1-tri-azeno)imidazole-4-carboxamide (DTIC) has resulted in the induction of antigenic specificities not found in parental cells. After the withdrawal of DTIC treatment, the antigenic sublines maintained the new properties indefinitely, as a heritable character although the mechanism of induction and the molecular nature of the new antigens are essentially unknown. Seven clones from the murine immunogenic leukemia L1210/DTIC were selected and studied in some detail. Three of the seven clones showed an increased immunogenicity in vivo since two clones (D5 and D7) were rejected by syngeneic mice and one (D6) prolonged the life span for 3 weeks (untreated tumours killed the mice in 7 days). The seven clones and the L1210/DTIC were recognized and lysed by in vivo primed, in vitro stimulated (with L1210/DTIC) lymphocytes. Therefore, the seven clones shared antigens with the L1210-DTIC immunogenic subline. Secondary stimulated lymphocytes to clone D5, and clone D7 were able to lyse D5, D6 and D7 cells, respectively, but were unable to recognize the remaining clones. From in vivo and in vitro studies, all the L1210/DTIC sublines are composed of cells carrying two or more distinct antigens. Each cell clone expressed one set of antigens. It was concluded that only a few antigens expressed in different cells were induced by a DTIC treatment of L1210 leukemia.

New antigenic specificities, not detectable on parental cells, have been induced by many investigators in mouse lymphomas by treatment with the antitumor agent 5(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC). The antigens are transmissible, after withdrawal of the drug treatment, as an inheritable character. The mechanism of induction, the molecular nature, and the number of the new antigenic specificities have not been completely elucidated. Four clones from murine leukemia L1210 isolated and expanded in vitro were treated in vivo with DTIC and the new sublines were studied in detail. The four drug-treated sublines studied exhibited strong immunogenicity since they were rejected by syngeneic animals. Immunosuppressed animals challenged with 10(7) A/DTIC or P/DTIC cells were reciprocally protected by the adoptive transfer of spleen cells from donors that had rejected a lethal challenge of A/DTIC or P/DTIC clone. In a similar fashion, the adoptive transfer of spleen cells obtained from animals that had rejected the Q/DTIC or the R/DTIC clones protected immunosuppressed mice challenged with Q/DTIC or R/DTIC cells. No antitumor activity was observed in cross-protective schedules other than those indicated. It was been concluded that (a) the L1210 leukemia line does not have antigenic cells, (b) four DTIC-treated clone sublines were rejected by compatible hosts, and (c) two mutually exclusive sets of antigens were expressed in four antigenic clone sublines.

A monoclonal antibody (L.1), reacting in vitro specifically with L1210 leukaemia cells in a complement-dependent cytotoxicity assay (CDC), has been exploited for serotherapy studies. Different regiments of L.1 treatment of CD2F1 mice bearing the semi-syngeneic L1210 leukaemia did not prolong the life span of tumor-bearing animals. Moreover, the administration of L.1 did not enhance the antitumour effects of cyclophosphamide. Studies of in vivo localization showed that L.1 was able to bind specifically to L1210 leukaemic cells, although 30-40% of the cells remained negative. The presence of L.1 in mouse blood was demonstrated up to 15 days after the inoculation. On the other hand, in vivo administration of L.1 was probably accompanied by loss of the cytotoxic activity, perhaps through a mechanism of complement inactivation, since the presence of undiluted normal mouse serum in a CDC assay inhibited the cytotoxic activity of L.1. Moreover, serum from L.1-treated mice did not display any cytotoxic activity, although the presence of the antibody could be demonstrated by indirect immunofluorescence. Shedding of the antigen defined by L.1 was probably not responsible for the failure of the serotherapy, since the L.1 neutralizing antigen could be found in body fluids only long after the start of therapy.

— It has been known that haemoatoporphyrin derivative (Hpd) has a preferential distribution in tissues with high mitotic index. Furthermore, cytocidal activity of light activated Hpd within the cells has been exploited in the therapy of experimental and human cancer. It is reported here that maximum, long lasting, although reversible, inhibition of DNA synthesis was obtained in Hpd-treated lymphocytes. However, Hpd-treated lymphoid cells did not stimulate allogeneic lymphocytes in the primary mixed lymphocyte reaction (MLR) culture. Phytohaemagglutinin (PHA) stimulated murine lymphocytes treated with Hpd and exposed to laser light have shown higher susceptibility to lysis than resting, PHA unstimulated, lymphocytes. In vitro DBA/2 stimulated C57 lymphocytes, inoculated into X-irradiated BD2F1 mice, upon Hpd treatment followed by exposure to light, did not cause a lethal graft vs. host reaction (GVHR). Haematoporphyrin derivative was preferentially incorporated by large and metabolically active cells. inhibited the incorporation of [3H]-thymidine in the lymphocyte nucleus and could be exploited to selectively remove blast cells from resting lymphocytes.

Photoradiation therapy is currently under investigation as a new form of treatment for solid malignant tumors in human. The method involves photosensitization of drugs and fluorescent dyes, such as Hematoporphyrin1. This dye has been shown to accumulate with a certain degree of specificity in tissues with a high mitotic index2. Moreover, neoplastic tissue might be identified by the fluorescence emitted from cancerous lesions that have incorporated the dye in larger amounts than normal tissues3. The photodynamic action has implicated singlet oxygen Ca short-lived excited molecular state of ground-state triplet oxygen) as the effective cytotoxic agent4.

We have studied the uptake of 125 I-labelled low-density lipoprotein (LDL) by seven experimental murine tumours in vivo. Four tumours (Lewis Lung carcinoma, B-16, MS-2 and Colon 26) showed a higher relative uptake of lipoprotein as compared to the liver, two (L-1210 and P-388) had a very low lipoprotein uptake, while lipoprotein uptake by tumour M5 was similar to that of the liver. The data was confirmed by tracing tissue uptake of lipoproteins using [ 14 C]sucrose-labeled LDL. These in vivo findings correlated well with the in vitro specific binding of 125 I-β-VLDL to membranes prepared from tumours, thus suggesting that the expression of the LDL receptor in the tumours is related to the in vivo uptake of lipoprotein. Further analysis of the LDL receptor by ligand blotting showed that the tumor receptor has several of the liver LDL receptor characteristics (including apparent M r , sensitivity to proteinases, and Ca 2+ requirement for lipoprotein binding). In summary, our data show that experimental murine tumours express the LDL receptor and suggest that the high relative in vivo uptake of LDL is determined by the elevated LDL-receptor expression in the tumours.

The cytocidal activity of light-activated hematoporphyrin derivative (Hpd) in experimental and human tumors is under investigation in many laboratories. This activity is based upon preferential incorporation of Hpd in malignant tissues and its photosensibilization by red light. Treatment of mice bearing MS-2 fibrosarcoma and B 16 melanoma, a metastatic tumor, with Hpd and laser light, externally or delivered through a quartz fiber optic imbedded directly into the tumor, significantly prolonged the median survival time. This therapy was compared with surgical excision of primary tumors, and preliminary results on metastatic neoplasm suggest that the photoradiation therapy is more effective than surgery.

The absorption spectrum of aluminum phthalocyanine with a mean degree of sulfonation of 2.1 (AlPcS 2 ), previously measured in vivo in a murine tumor model (L1210 leukemia), showed a significant (10 to 15 nm) red shift with respect to the spectrum in solution and in vitro. To investigate whether this behavior was due to the peculiar model considered (ascitic tumor), the absorption line shape was evaluated in vivo noninvasively in a solid tumor (murine MS-2 fibrosarcoma) by means of time-resolved reflectance. The spectrum acquired in the range of 650 to 695 nm after the injection of 2.5 or 5 mg/kg body weight AlPcS 2 confirms the red shift to 680 to 685 nm. Experiments performed ex vivo suggest that the spectral shift is related to the interactions of the drug with the extracellular biological substrate and depends on the sensitizer concentration.

Mice bearing the MS-2 fibrosarcoma were administered 0.25, 0.5 or 1 mg kg(-1) body weight (b.w.) of sulphonated aluminium phthalocyanine (AlS(2)Pc) (with average degree of sulphonation of 2.1), and time-gated fluorescence images were acquired up to 6 h after the injection. Different excitation wavelengths (610, 650 and 670 nm) were tested. Red light excitation and 3 ns delayed detection allow one to minimize natural fluorescence and scattered laser light, respectively. The best conditions for tumour detection are reached under either 650 or 670 nm Excitation, 2-4 h after the administration of either 0.5 or 1 mg kg(-1) b.w. of AlS(2)Pc. In these situations, the average fluorescence contrast between tumour area and surrounding healthy tissue is2, providing a clear identification of the pathological region. However, tumour localization is possible even after the injection of 0.25 mg kg(-1) b.w. of sensitizer. In conclusion, under low power excitation (100MuW cm(-2)), the technique allows real time detection of an intradermal tumour with good contrast.

Photodynamic therapy (PDT) is systemic administration of tumor localizing photosensitizers and subsequent irradiation with light of the appropriate wavelength. The combination of drug uptake in malignant tissues and selective delivery of laser-generated light provides effective therapy, with efficient tumor cytotoxicity and minimal normal tissue damage. There have been few studies of the effects of photoactivated photosensitizers on the host immune response. Since immunity is important in the control of tumor growth and spread, we have examined, in our laboratory, the effects of photoactivated phthalocyanines on the antitumor immune response. Immunosuppressed and normal mice bearing the MS-2 fibrosarcoma treated with 5 mg/kg of aluminum disulfonated phthalocyanine (AIS2Pc) and then the tumor mass exposed to laser light (100 mW/cm2 x 10 min) or treated with surgical excision of the tumor survived indefinitely, with no difference between the different groups. The survivors, tumor-free 100 days after the treatment modalities described above, were rechallenged with the parental MS-2. Some groups of surviving animals were immunosuppressed with cyclophosphamide before the injection of the tumor. Resistance to rechallenge was evidenced only in normal surviving animals cured by PDT, while the immunodepressed surviving animals and animals cured by surgery died of tumor. Finally, mice, cured by PDT and tumor-free, rechallenged with L1210 and P388 murine leukemias did not survive. These results suggest that a potential and specific 'antitumor immunity' is induced by PDT with photoactivated AIS2Pc.

Time-resolved reflectance was used to measure the absorption spectrum of hematoporphyrin derivative (HpD) in vivo in a murine tumor model. Reflectance measurements were performed in the 600-640 nm range on mice bearing the L1210 leukemia. Then the animals were administered 25 mg/kg body weight of HpD intraperitoneally. One hour later the reflectance measurements were repeated. Fitting of the data using the diffusion theory allowed assessment of the absorption coefficient before and after the administration. As a difference between the latter and the former data, the in vivo absorption spectrum of HpD was evaluated. Maximum absorption was measured at 620-625 nm. Similar spectral behavior was obtained for HpD in solution in the presence of low-density lipoproteins.

The absorption properties of photosensitizers for the photodynamic therapy of tumors (PDT) may be significantly influenced by their interactions with the biological substrate, leading to an in vivo spectrum, which cannot be predicted on the basis of in vitro studies. Therefore, for the development of an optimized clinical protocol, it is fundamental to estimate the absorption spectrum in vivo non-invasively, thus determining the best irradiation wavelength. This measurement can be performed by means of time-resolved reflectance, which allows to evaluate both the absorption and the scattering coefficients, and therefore provides also information on the light penetration in tissues, useful to define the optimal light dose for therapeutical purposes.

A system for time-gated fluorescence imaging was used to perform measurements on tumor-bearing mice treated with hematoporphyrin derivative (HpD). The aim of the study was to define the potential of this technique in the diagnosis of tumors by taking advantage of the long fluorescence lifetime of the exogenous dye with respect to the decay times of the natural fluorescence. After the administration of three different drug doses (5, 10 and 25 mg/kg body weight), fluorescence images were acquired at various uptake times (from 2 h to 10 d), to determine the best instrumental conditions and experimental procedure for the detection of tumors in the murine model considered. The optimal fluorescence contrast between the tumor area and the surrounding healthy tissue was found at 12 h after the administration of either 5 or 10 mg/kg HpD and was anticipated at 8 h for the highest drug dose. In this optimum condition, the tumor region could be identified even after the injection of 5 mg/kg HpD. A better fluorescence contrast was always obtained in 15 ns-delayed images with respect to synchronous ones.