Combination of photodynamic therapy + immunotherapy + chemotherapy in murine leukiemia.

Photodynamic therapy (PDT) is a treatment for cancer based on the photosensitization of tumor cells by photosensitive drugs and their subsequent destruction on exposure to light of particular wavelength. The combination of drug uptake in malignant tissues and selective delivery of laser-generated light provides an effective therapy with efficient tumor citotoxicity and minimal normal tissue damage. Since immune response of the host is important in the control of tumor growth and spreading, PDT is able to increase the antitumor immunity. In our laboratory we examined the antitumor effect of combination of PDT, with photoactivated M-THPC (meta-tetrahydroxyphenylchlorin, FOSCAN, Temoporphirin), adoptive immunotherapy, with immune lymphocytes, and chemotherapy on advanced murine tumors. Mice bearing L1210 tumor were treated at day +4 with Navelbine (NVB 1mg/Kg), at day +5,+6 with PDT (0.3mg/Kg of mTHPC and 100mW/cm(2) x 200'' of exposure of laser light), and at day + 7 with immune lymphocytes(IL), collected from mice pretreated with PDT(2x10(7) cells). The results show that the combination NVB + PDT + IL demonstrates a significant synergistic antitumor effect while the chemotherapy treatment with low dose of the drug is uneffective. The same positive results were obtained with the combination of Cisplatin (CDDP 0.5mg/Kg), PDT and IL, while the CDDP treatment alone is completely uneffective. In conclusion, these results suggest that it is possible to completely cure animals bearing advanced tumors, with a combined therapy, PDT + adoptive immunotherapy + low dose chemotherapy.

Cell survival under nutrient stress is dependent on metabolic conditions regulated by Akt and not by autophagic vacuoles.

Akt activation assists tumor cell survival and promotes resistance to chemotherapy. Here we show that constitutively active Akt (CA-Akt) cells are highly sensitized to cell death induced by nutrient and growth factor deprivation, whereas dominant-negative Akt (DN-Akt) cells have a high rate of survival. The content of autophagosomes in starved CA-Akt cells was high, while DN-Akt cells expressed autophagic vacuoles constitutively, independently of nutrition conditions. Thus Akt down-regulation and downstream events can induce autophagosomes which were not directly determinants of cell death. Biochemical analysis in Akt-mutated cells show that (i) Akt and mTOR proteins were degraded more rapidly than the housekeeping proteins, (ii) mTOR phosphorylation at position Thr(2446) was relatively high in DN-Akt and low in CA-Akt cells, induced by starvation in mock cells only, which suggests reduced autoregulation of these pathways in Akt-mutated cells, (iii) both protein synthesis and protein degradation were significantly higher in starved CA-Akt cells than in starved DN-Akt cells or mock cells. In conclusion, constitutively active Akt, unable to control synthesis and wasting of proteins, accelerates the death of starved cells.

Immunotherapy of bilateral lymph node metastases in guinea pigs by intralesional or paralesional injection of Mycobacterium bovis (BCG).

Bilateral axillary lymph node metastases occurred after intradermal (id) injection of line 10 hepatocellular carcinoma cells over the thoracic spine of inbred guinea pigs. Excision of the dermal tumor 7 days after injection of tumor cells did not prevent the development of metastases. Injection of BCG into dermal tumors without surgery led to their regression and prevented the growth of microscopic metastases in both right and left superficial distal axillary lymph nodes. Bilateral id injection of BCG between the dermal transplant and each of the regional lymph nodes followed by excision of the dermal tumor also prevented progression of metastases. Unilateral id injection of BCG before excision of dermal tumors failed to retard metastases in contralateral superficial distal axillary lymph nodes. These results suggested that elimination of microscopic lymph node metastases required delivery of adjuvant to or near each metastatic site. Systemic tumor immunity alone may be inadequate to eradicate lymph node metastases.

Chemotherapy and immunotherapy of L1210 leukemic mice with antigenic tumor sublines.

Tumor cells, treated in vivo with anticancer compounds, may acquire new antigenic specificities in addition to any original antigens associated with parental tumors. Treatment of mice carrying the parental leukemias L1210 Ha or L1210 Cr with leukemia cells antigenically altered by treatment with 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (L1210 Ha/DTIC and L1210 Cr/DTIC, respectively) was essentially ineffective in prolonging the life span of the animals. However, synergic therapeutic activity was exhibited by administration of L1210 Ha/DTIC cells plus 1,3-bis(2-chloroethyl)-1-nitrosourea in the treatment of the moderately immunogenic L1210 Ha leukemia and by the combination of L1210 Cr/DTIC cells and lymphocytes immune to L1210 Cr/DTIC administered with 1,3-lymphocytes immune to L1210 Cr/DTIC administered with 1,3-bis(2-chloroethyl)-1-nitrosourea in the treatment of the low immunogenic L1210 Cr leukemia. Early and advanced L1210 Cr-bearing mice showed marked increases in survival time and a significant number of tumor-free survivors on treatment with cyclophosphamide followed by transfer of lymphocytes immune to L1210 Cr/DTIC cells. When parental tumor cells were used as the immunogen, the therapeutic effect was diminished. Thus, in the current investigation, although immunotherapy per se was essentially ineffective, the immunochemotherapeutic modalities used were successful in markedly increasing the survival time of leukemic animals and resulted in an incidence of cures.

Hematoporphyrin derivative rescue from toxicity caused by chemotherapy or radiation in a murine leukemia model (L1210).

Hematoporphyrin [1,3,5,8-tetramethyl-2,4-bis(hydroxyethyl)-porphin-6,7-dipropio nic acid dihydrochloride derivative] (HPD) is a compound that was studied in a number of laboratories because of its cytocidal activity after activation by light. Modification of immune function seen during the photochemotherapeutic studies prompted attempts to determine the effect of HPD on the immune and hemopoietic systems. Splenic hyperplasia as well as marrow hypercellularity were noted in mice treated with HPD. In vitro phytohemagglutinin or lipopolysaccharide stimulation of spleen lymphocytes caused normal or scant increases in blast transformation compared to the stimulation index for lymphocytes from untreated animals. HPD treatment did not significantly alter production of antibody to sheep red blood cells, as evaluated by hemagglutination or hemolytic assay. In contrast, HPD treatment did promote an increased number of spleen colonies in lethally irradiated mice transfused with syngeneic bone marrow. The capacity of HPD to increase the number of bone marrow and spleen cells has been exploited to accelerate the recovery from peripheral leukopenia induced in animals by previous drug or radiation treatment. The time for full return from severe leukopenia induced by an antimetabolite compound (5-fluorouracil) or an alkylating agent (cyclophosphamide or X-rays was significantly shorter in mice treated with HPD than in controls. Furthermore, improved survival was demonstrated in irradiated mice after HPD treatment. Finally, HPD treatment of L1210 leukemic mice did not affect the antitumor activity of cyclophosphamide. If the properties described here be confirmed, HPD might contribute to recovery of leukopenic cancer patients.

A bcl-2/IgH antisense transcript deregulates bcl-2 gene expression in human follicular lymphoma t(14;18) cell lines.

The 14;18 chromosome translocation, characteristic of most human follicular B-cell lymphomas, juxtaposes the bcl-2 gene with the IgH locus, creating a bcl-2/IgH hybrid gene. By mechanisms that are still under investigation, this event increases the cellular levels of the bcl-2 mRNA and thereby induces an overproduction of the antiapoptotic BCL-2 protein which is likely responsible for neoplastic transformation. In an effort to identify potential upregulators of bcl-2 activity in t(14;18) cells, we found, by strand-specific RT-PCR, a bcl-2 antisense transcript that is present in the t(14;18) DOHH2 and SU-DHL-4 but not in the t(14;18)-negative Raji and Jurkat lymphoid cell lines, and thus appears to be dependent on the bcl-2/IgH fusion. This antisense transcript is a hybrid bc1-2/IgH RNA, that originates in the IgH locus, encompasses the t(14;18) fusion site and spans at least the complete 3' UTR region of the bcl-2 mRNA. To achieve some insight into its biological function, we treated the t(14;18) DOHH2 cell line with oligonucleotides (ODNs) by specifically targeting the bc1-2/IgH antisense strand. These ODNs lowered bcl-2 gene expression, inhibited neoplastic cell growth by inducing apoptosis. We would like to propose the hypothesis that the bc1-2/IgH antisense transcript may contribute, by an unknown mechanism, to upregulation of bcl-2 gene expression in t(14;18) cells. The possibility has been considered that the hybrid antisense transcript mask AU-rich motifs present in the 3' UTR of the bcl-2 mRNA characterized in other genes as mRNA destabilizing elements.

Assimilation of LDL by experimental tumours in mice.

We have studied the uptake of 125I-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 [14C]sucrose-labeled LDL. These in vivo findings correlated well with the in vitro specific binding of 125I-beta-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 Mr, sensitivity to proteinases, and Ca2+ requirement of 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.

Rapamycin increases the cellular concentration of the BCL-2 protein and exerts an anti-apoptotic effect.

The immunosuppressant rapamycin, an immunophilin-binding antibiotic, has been studied in follicular B-cell lymphoma lines that express the highest level of the BCL-2 protein. The growth rate of human follicular B-cell lymphoma lines was slowed more efficiently than that of other human B-cell lines or non-B-cell lines. This effect was dependent on the arrest of cells in the G(1) phase; the number of apoptotic cells was not increased. Rapamycin inhibited apoptosis or caspase activation induced by cytotoxic drugs, whereas caspase activation by doxorubicin was not inhibited. The increase in the cellular concentration of BCL-2 protein was related to its concentration in the steady state and was unrelated to the amount of bcl-2 mRNA. The increase of BCL-2 level in the cells rather than its level in the steady state may be important for drug resistance. The biochemical target of rapamycin, the mTOR kinase, may be a candidate sensitising agent for chemotherapy. This effect of rapamycin shows that G(1) arrest and protection from apoptosis are combined events susceptible to regulation by pharmacological means.

B16 melanoma response in vivo to photochemotherapy with mitoxantrone and red light.

The cytocidal activity of light-activated mitoxantrone in mice bearing B16 melanoma was investigated. Mice inoculated with 10(6) tumor cells on day 0 were i.p. injected with 1 mg/kg body weight of mitoxantrone on days 1, 5 and 9 and exposed to 108 J/cm2 of suitably filtered red light from a halogen lamp on days 2, 6 and 10. The treatment significantly prolonged the median survival time compared to both therapy with mitoxantrone and with red light alone.

Efficacy of photodynamic therapy against doxorubicin-resistant murine tumors.

Photodynamic therapy (PDT) is a relatively new cancer treatment modality that employs light excitation of a photosensitizer to yield cytotoxic oxygen-related species. In the present study we explored whether PDT would have therapeutic effect against doxorubicin-resistant murine tumors. We compared the efficacy of PDT with aluminium disulphonated phthalocyanine (A1S2Pc) and laser light on the doxorubicin-sensitive murine tumors, B16 melanoma (B16), L1210 leukemia (L1210), P388 lymphoma (P388) and the corresponding doxorubicin-resistant lines (B16/Dx, L1210/Dx and P388/Dx). Mice bearing L1210-L1210/Dx, P388-P388/Dx and B16-B16/Dx, were treated with 5 mg/kg of A1S2Pc and laser light (100 mW/cm2 x 10 min of exposure) or with doxorubicin (10 or 12 mg/kg i.v.). The results show that PDT is active versus all tumors while doxorubicin is effective only against the three sensitive tumor lines (L1210, P388 and B16). These observations suggest that PDT might be a beneficial alternative treatment for drug-resistant tumors.

Hematoporphyrin derivative photoradiation therapy in murine solid tumors.

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 B16 melanoma, a metastastic 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.

In vivo assimilation of low density lipoproteins by a fibrosarcoma tumour line in mice.

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.

Antitumor efficacy of the combination of photodynamic therapy and chemotherapy in murine tumors.

Photodynamic therapy (PDT) is based on the administration of tumor-localizing photosensitizers followed by light exposure of the tumor mass. The photocytotoxic effects are mainly caused by the generation of singlet oxygen. Recently, PDT has been proposed for use in combination with anticancer chemotherapy with a view to exploiting any additive antitumor effect. We investigated the effect of PDT with photoactivated aluminum disulfonated phthalocyanine (AlS2Pc) combined with the antiblastic drugs Adriamycin (ADR) and cisplatinum (CDDP) on murine tumors. Mice bearing L1210 leukemia and P388 lymphoma were treated with ADR or CDDP and subsequently treated with PDT. Low chemotherapy doses were ineffective, but the combination of antiblastic drugs + PDT had a significantly additive antitumor effect. In conclusion, with this combined therapy we were able to greatly reduce the effective doses of antiblastic drugs, thus lowering their toxic effects on normal host tissues.

Comparative study of the therapeutic effect of photoactivated hematoporphyrin derivative and aluminum disulfonated phthalocyanines on tumor bearing mice.

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').

Epithelial Membrane Antigen. Its use in the cytodiagnosis of malignancy in serous effusions.

The cytological distinction between reactive mesothelial and malignant cells frequently causes problems for the diagnostic cytologist. In order to determine whether an immunocytochemical method might help resolve these difficult cases, we have stained smears from 309 serous effusions from 246 patients for the Epithelial Membrane Antigen (EMA). The EMA staining was classified as strong, weak or negative. Carcinoma cells (as diagnosed by conventional cytology) stained strongly for EMA in 63 of the 116 positive smears (54%). Five out of 15 (33%) of cytologically suspicious smears from patients with known carcinomas gave a strong EMA stain. Of particular interest were three effusions in which malignant cells were not identified in conventionally stained smears and in which a small number of EMA positive cells were identified. The EMA positive cells were subsequently restained by the Papanicolaou method and identified as malignant on retrospective morphological examination.

Time-gated fluorescence imaging for the diagnosis of tumors in a murine model.

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.

Fluorescence lifetime imaging of experimental tumors in hematoporphyrin derivative-sensitized mice.

Tumor detection has been carried out in mice sensitized with hematoporphyrin derivative (HpD) by measuring the spatial distribution of the fluorescence lifetime of the exogenous compound. This result has been achieved using a time-gated video camera and a suitable mathematical processing that led to the so-called "lifetime images." Extensive experimental tests have been performed on mice bearing the MS-2 fibrosarcoma or the L1210 leukemia. Lifetime images of mice show that the fluorescence decay of HpD is appreciably slower in the tumor than in healthy tissues nearby, allowing a reliable detection of the neoplasia. The lengthening of the lifetime in tumors depends little on the drug dose, which in our experiments could be lowered down to 0.1 mg/kg body weight, still allowing a definite tumor detection. In order to ascertain the results achieved with the imaging apparatus, high-resolution spectroscopy, based on a time-correlated single photon counting system, has also been performed to measure the fluorescence lifetime of the drug inside the tumor and outside. The outcomes obtained with two techniques are in good agreement.

Absorption spectrum of hematoporphyrin derivative in vivo in a murine tumor model.

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.

Fluorescence imaging during photodynamic therapy of experimental tumors in mice sensitized with disulfonated aluminum phthalocyanine.

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 AlS2Pc 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 AlS2Pc absorption spectrum shifts toward shorter wavelengths as a result of the irradiation.

Toxicity of fenclor 42 in mice: effects on immunocompetent cells.

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.