PERSPECTIVES

FOR

THE COMBINED USE OF PHOTODYNAMIC

THERAPY

AND HYPERTHERMIA IN CANCER PATIENT

I. Freitas*, McLaren***

P.

Pontiggia**,

G.F.

Baronzio**,

J. R.

*Laboratorio di Anatomia Comparata, Universita di Pavia, Pza Botta 10, 27100, Pavia, Italy. ** Dept. of Hematology Oncology, Clinica Citta di Pavia, Via Parco Vecchio 27100, Pavia, Italy. *** Emory University School of Medicine, Atlanta, Georgia, USA Chemotherapy and/or radiotherapy widely used in the last decades for cancer treatment are frequently barely effective on tumor growth and metastatic spread. Years of disappointing results, at least for the large majority of human solid tumors, induced the search for more active treatments. Photodynamic therapy (PDT), a relatively new method, has been tested for the treatment of a certain number of chemoresistant cancers, sometimes successfully (1). Recent in vitro and in vivo experiments suggest that the combination of hyperthermia (HT) and PDT can increase the therapeutic effect of these two therapies when used in combination (2,3,4). The presence of molecular oxygen is essential for the success of PDT, since single oxygen and other oxygen-derived species are the main cytotoxic agents in this treatment modality. When the oxygen concentration in the medium is low, the damaging effect of photosensitization on cells is remarkably reduced or even nil. This raises serious problems in the clinical use of photosensitization for treatment of cancer, since most solid tumors contain cells living under extremely low oxygen pressures. To understand fully the extent of protection from PDT that hypoxia confers on tumors cells, the following points should be considered: (a) the oxygen concentration in the air at sea level is about 0.24 mM and it's partial pressure is 152 mmHg: (b) in arterial blood, the oxygen concentration is about 0.12 mM (78 mmHg); (c) in normal tissues p(O ) of about 40 mmHg are f 0 u n d (63. 1 mM); ( d) i n tum 0 r s 0 < rf( 02) .;: 4 0 mmHg has bee n determined. The number of cells living and in some cases proliferating in p(02) values lower than 5 mmHg (7.89 mM), i.e. the "hypoxic fraction", varies widely with tumor type and growth site. Hypoxic cells may reoxygenate after the destruction of oxygenated cells by therapy and resume active proliferation, thus causing local tumor growth. Consensus on Hyperthermia jor Ihe 1990s Edited by H. I. Richer el af. Plenum Press, New York, 1990

511

It was observed that if PDT is immediately followed by HT, a superadditive (synergistic) potentiation is obtained. Some hypothesis have been put forth, but a satisfactory explanation for such synergism has not been proposed. In this work, through analysis of various aspects of the two therapies is performed and a possible interaction mechanism is derived. This analysis moreover explains the specific treatment sequence requested to obtain a synergistic potentiation (that is, PDT prior to, and not vice versa). MECHANISM OF ACTION AND EFFECT ON TUMOR VASCULATURE PDT is primarily based on the preferential uptake and retention of some porphyrins by tumor (and, in general)by all tissues characterized by a high proliferation rate with respect to most normal tissues. A differential porphyrin distribution is observed at tumor level: the extracellular compartment and, in particular, the vascular area, macrophages and necrotic areas contain higher drug concentrations than the tumor cells themselves(5). (N,B.The vascular endothelial cells of tumors,where the highest percentage of drug concentration is found, have a proliferation rate that exceeds what is found in normal tissues by a factor ususally greater than 20). After specific light activiation of the porphyrin-containing tumor, a highly cytotoxic species,singlet oxygen,is produced due to porphyrin sensitized photodynamic action. Several hours after PDT tumors show evident hemorrage,congested vessels, platlet thrombi and lysed tumor cells. The main factor that contributes to tumor destruction by PDT seems to be the stoppage of blood flow and the resultant ischemia of the tumor due to collapse of the vasculature(6).When HT is performed before PDT, the vascular modification caused by heat drastically decreases light penetration in the tumor; the efficiency of PDT is therefore highly impaired. MICROENVIRONMENTAL OXYGEN AND pH RELATED EFFECTS Tumors usually contain a high proportion of hypoxic cells that may be resistant to PDT (7).The hypoxic cells, due to their high glycolisis, live in an acidic environment, and therefore are very heat sensitive. There may thus be a complimentary effect of HT and PDT, in terms of the destruction of this population, such as the one that exists between HT and Radiotherapy (8). PDT causes a sharp reduction of P02, due to the abrupt reduction for blood flow, and no significant shift of PH, since it does not evoke any metabolic stimulation (9). If HT is performed after PDT on already badly perfused environment ulteriorly hinders the efflux of heat from the tumor;the temperature differential between the tumor and the surrounding, well perfused, normal tissues will therefore ultimately increase. This may be a further parameter contributing to the synergism between PDT and HT, and again to the observed sequence importance. The increase in acidity and the

512

decrease in nutritional supply during HT increases the thermal damaging effect on the survivihg tumor cells. An effect that has not so far deserved due attention is the influence of the heat-induced vascular alterations on the normal tissue and to the tumor. As a matter of fact, the increase inblood flow and in vascular permeability observed concentration therejon the other hand,vascular status in the tumor may decrease drug de1ivery,therefore decreasing the therapeutic index(7). CELLULAR RELATED EFFECTS The macroscopical effects of PDT and HT on the vascu1ature,presumably the main responsible for the observed synergism,are due to collapse of the tumor peculiar endothelia (10). The microscopical effects that condition the response not only of the tumor endothelial cells but also of macrophages and the tumor cells themselves, worth therefore to be considered. The cellular damaging effects of PDT and HT will now compare recalling that cell activity requires selectivity of membrane permeability, and that reproductive capability is assured by the integrity of the nucleus and of the intracellular organelles. Effect of PDT The cellular response to PDT is dependent on several experimental parameters (e.g. composition,concentration and mode of delivery of the sensi1tizer, incubation time, oxygen concentration, photoactivatin energy, presence of serum proteins(11). Furthermore, cytotoxicity is due to the unspecified action of free radicals, that are produce after attack of singlet oxygen to electron rich areas of biomo1ecu1es (e.g. conjugated double bonds). It is thus rather difficult to evaluate the initial lesion, since free radicals initially formed in a well defined localization soon initiate chain reactions spreading damage allover the cell. Electron micropsy, EM, is an almost ideal technique to observe damage onset and spreading. Although some papers describe the EM picture of a cell after PDT, they refer to different experimental conditions. One, however, deserves mentioning since it describes not only damage but also the structural modifications that suggest an attempt of repair. (12). The main effects observed are: (a) proportionally, much less proliferating and more degenerating and dying cells than before PDT; (b) soon after PDT,enlargement of cellular interspace, reduction of cell volume, diminution and shrinkage of microvilli and appearance of pseudopodiaj(c)part of the membrane destroyed with cytoplasm shed outsidej (d) vacoules of different dimensions, the larger of which occupy almost half of the ce11j (e) loose atypical and no more compact, filaments in the cytop1asmj (f) immediately after PDT, blurred mitochondrial membranes, some shrunk mitochondria with indistinct cristae filled with

513

high density material and vacuoles, some electron density particles; as the time after PDT increases, fewer particles and more vacuoles in the mitochondria; (g) rough endooasmatic reticulum, RER, filled with high density material (h) formation of an increased number of Golgi complexes connected to several vacuoles; (i) synthesis of new lysosomes that pile up in the concave part of the nucleus; (l)rounded up nuclei with decrease electron density and decreased granular euchromatin; heterochromatin attached to the nuclear membrane; (m) several mitosis containing degenerated mitochondria,dilated ER vacuoles;(n) centrioles and condensed chromosome still seen in the mitosis without spindle fibers.Membranes, and, in particular, the plasma membrane, are the most important cellular targets of PDT.The mainmolecular effects of PDT at membrane level are: oxidation of unsaturated liquids and cholesterol, and (ii) crosslinking and oxidation of some -SH groups of proteins. These chemical alterations lead in turn to a marked inhibition of the physiological activities of the membrane, and, in particular, of passive and active transports; this effect shows a marked correlation with porphyrin-induced loss of cell viability(13). It was observed, in particular:(a) enhancement of ANS binding, evidentiating increased membrane hydrophobicity;(b) inhibition of thymidine,nucleoside and main acid transport,that in part explains the inhibition of DNA and protein synthesis;(c)increased permeability to chromate, to K (in pat due to the oxidation of the -SH groups), and to actinomycine 0; (d) inhibition of membrane enzymes (e.g. 5nucleotidase, alkaline phosphatase, Na-K ATPase); (e) inhibition of cappine of antibodies and CON A agglutionation, which are properties that require normal fluidity of the plasma membrane and integrity of cytoskeleton; and (f) that cholesterol impregnation of leukemic cell membranes induces resistance to PDT. It should be mentioned that the cellular structures surrounded by membranes communicate dynamically with each other apparently by means of a continuous interchange in membrane fragments. Therefore, peroxidation initiating at the plasma membrane may easily propagate to other membranuous compartments. Mitochondria are one the POTs main targets: Essential functions (e.g. respiration, oxidative phosphorilation and Ca membrane transport)are affected since the activity of enzymes such as cytochrome C oxidase and succinate dehydrogenase, and ATPase is a well known effect of photo sensitization that ultimately causes the release of hydrolytic enzymes. After PDT, leakage of acid phosphatase, B-glycoronodase, Arylamidase, B-galacrosidase and B-N-acetylglucosdaminidase was indeed reported. In particular, damage to the lysosome of endothelial cells after porphyrin sensitization in vivo causes stasis of blood, aggregation of cells, intravascular hemolysis and irreversible damage. Isolated DNA can be photodynamically damaged by porphyrins: alkali-labile sites and single and double strand breaks were reported. Photodynamic crosslinking of DNA to protein is also induced. When whole cells are submitted to PDT,DNA sina 1q strand breaks also

514

occur,at significantly lower rates than than with x-rays. The chemical nature of the strand breaks induced by PDT seem to be different than those produce by x-rays. These lesions are not always easily repaired,particularly when high light doses are used. The ability of DNA to serve as a template to DNA dependent RNA synthesis is impaired. Crosslinking of DNA proteins is also observed with whole cell irradiation. As concerns the repair processes after PDT, the following observations were made:(a)prophyrin sensitization rapidly inhibits viral and mammalian DNA polymerasees; (b) cells are able to accumulate and recover from PDT damage in a way similar to the accumulation and recovery from x-ray damage, "Elkind repair"; and, (c) PDT is followed by synthesis of stress proteins, similar to heat shock proteins or to the protein synthesized in response to glucose deprivation and chronic anoxia. These morphologic and biochemical effects were extensively reviewed (14). The sensitivity to photodynamic inactiviation increase as the cells progress in their cell cycle from Gl to mid S phase (15). The particular susceptibility during the S phase was attributed to a peculiar condition of reduced microviscosity of plasma membrane damage in this phase. Although the speculation about a possible correlation between sensitivity to PDT and membrane viscosity seems quite plausible, another fact deserves consideration: in the S phase the DNA molecule is in a conformation fully extended for replication,and therefore particularly vulnerable to attack by external agents. These agents might be, for instance, the hydrolytic peroxidation. The effects of PDT on the nuclear structures do confirm the vulnerability of DNA, but it has not yet been clarified how the damige to the nucleus depends of the cell cycle phase. The damage bought to the DNA molecule may hinder its ability to act as a template for RNA synthesis, therefore impairing the synthesis of proteins with repair function. Effects Qf Hyperthermia An EM study that very exhaustively depicts the onset and propagation of damage induced by HT was published years ago (16). It was observed in this work that, during the first few days after treatment, the cellular morphology of nonmalignant tumor cells (e.g. fibroblasts and endothelial cells) was only slightly and reversibi1y damaged, while the ma1iqnant cells showed massive destructive alterations. This- study also points out that the presence of a hypertrophic Golgi area,contemporary to the formation of larger lysosomes, indicates that a high lysosomal activity may be primarily important in heat induced cytotoxity. In the EM study above reported, concerning the study of a tumor in vivo the thermal effect on the endothelial cells was irrelevant with respect to the effect on the malignant cells and, in particular, reversible. However, since HT is followed by occlusion of blood flow, damage to the vasculature should also play an important role in tumor

515

control. In effect, it was recently reported that, in vitro, the capillary endothelial cells are more heat sensitive than fibroblasts; at temperatures normally used in HT,damage results in cell death, demonstrable already at 30 min after initiation of a 44 C/30 min dose. In particular, capillary endothelial cells stimulated to proliferate are even more heat sensitive than quiescent endothelial cells. Since the tumor vasculature contains a significantly higher proportion of proliferating endothelial cells than the normal vasculature, the enhanced thermal sensitivity of the tumor neovasculature with respect to that of the host tissue is easily explained(6).The heat induced lethality results from the combined effect of several lesions to key structures in the cell, as it happens with PDT. For the sake of clarity, they will be again presented separately. The plasma membrane, which is the first cellular structure where heat energy is deposited, contains lipids and proteins highly susceptible to thermal damage. The membrane morphology after HT, observed with scanning EM, shows a rapid and irreversible transformation: from ordinarily smooth it acquires a cobblestone morphology; most microvilli disappear and those that remain clump into a cap(I?). It was long supposed that cytotoxicty was due to an irreversible increase in membrane fluidity, but it was later demonstrated that there is no correlation between thermal cell killing and membrane liquid fluidity (18). On the other hand, the activiation energy required for cell killing indicated a protein target for thermal damage. Heat has moreover a differential effect on the Na-K-ATPase: the capacity to bind ouabain decreases, while the ATP hydrolyzing acitivity on the membrane inner surface retains most of the original acitivity (20). This shows that, within the same protein that acts as sodium-potassium pump, two different regions are deputed to the mentioned properties, and each of these is influenced differently by heat. One of the main mechanism also proposed for cell ktlling by HT consists in the cell digestion by hydrolytic enzymes released after lysosome rupture. This assumption is based on the synthesis of an increased number of lysosomes, and of an increased proteolytic enzyme activity after HT (21).Moreover, tumor cell lysosomes seem more heat-labile than normal cell lysosomes. However:(a) neither lysosomal damage by photosensitization with neutral red, nor intralysomial inhibition of hydrolases by trypan blue, modifies the onset of inhibition of respiration of tumor cells by HT(22);and,(b) agents that modify the lysosome membranes, either by labilizing them (retinol) or by stabilizinp them (hydrocortisone), do not alter the response of the cells to HT(23). These experiments indicate that lysosomal damage by itself may not cause cell death, although it may substantially contribute to it. The proteins of the mitochondrial membrane undergo structural transitions at hyperthermic temperatures. The marked morphological alterations of mitochondria, that are

516

illustrated by EM, have a functional equivalent as inhibition or depression of the oxidative metabolism (24) with consequent decrease in ATP levels(25). Anaerobic glycolysis in tumors, on the other hand, seem not to be very affected by heat, and thus represents the only energy producing pathway in heated cells. Glucose deprivation, in effect, highly increases the heat induced toxicity(26). The increased importance of glycolysis causes an increase in acidity in tumor cells and in extracellular space in the tumor tissue. In these conditions, the activity of the lysosomal acid hydrolases may be intensified. It was already mentioned that heat affects the number and distribution of the "primary stimulus" receptors of the cell located in the plasma membrane. Heat besides affects "secon message" substances, such as Ca t !-, Ht-, and cyclic AMP, increasing their intracellular concentration. It was suggested that the increase in cell volume during and after HT is due to osmotic changes resulting from H ion flux(27). A recent speculation, moreover, proposes that HT induces alterations in the information flow through the cell, that starts at the outer receptors of the plasma membrane,continues in the internal signal effectors and later disrupts the cytoskeleton and inhibits protein and DNA synthesis(25).Therefore, heated cells are unable to complete cytokinesis, they become tetraploid, and loose their reproductive integrity(28). CONCLUSIONS In spite of these premises, most researchers involved in PDT clinical practice have not yet faced the fact that tumor regrowth due to spared hypoxic cells can easily be foreseen and should thus be expected after a single PDT dose. Since this event seriously hampers PDT and since established procedures are readily available to overcome hypoxia, clinicians should not ignore the interest and potentiality of the association of such methods with PDT in clinical experimentation. A detailed analysis of the biological effects of PDT and HT explains why this combination should be complementary if PDT is performed before HT. In effect, while PDT affects the tumor vasculature in the first place, and only secondarily the well-oxygenated tumor cells, HT primarily destroys the tumor cells living in an acidic, nutritionally deprived, environment (that are, moreover, hypoxic), and, to a lesser extent, also the tumor vasculature. If HT is performed before PDT, a synergism is not obtained, since HT causes a sharp decrease in tumor oxygenation (oxygen is one of the PDT"fuels") capillary collapse and hemorrhagic phenomena, that hinders light penetration in the tissues. On the contrary, PDT before HT favors HT; in effect, the deterioration of the tumor blood flow induced by PDT further hinders heat dissipation by blood circulation, and therefore increases the temperature differential between the tumor and the normal surrounding tissue. Also at the cellular level the damaging effects PDT and HT reinforce each other, particularly at membrane

0*

517

and nuclear levels. The overall mutagenicity of the combination PDT+HT, not yet determined, may not be negligible, although, separately, each of these therapies seems to be much less dangerous than equivalent doses of xrays. Particular caution is requested to this specific point. REFERENCES 1) T.J. Dougherty:Photodynamic Therapy. In G.YORI and C.PERRIA eds, photodynamic Therapy of Tumors and Other Diseases. Libreria Progetto, Padova,1985 Dougherty: 2) S.M. WALDOW, B.W. HENDERSON, T.J. Potentiation of Photodynamic Therapy by Heat: Effect of Sequence and Time Interval Between Treatments in Vivo. Lasers. Surg. Med. 5:83-94, 1985 3) T.S. Mang, T.J. Dougherty: Time and Sequence Dependent Influence of In Vivo Photodynamic Therapy Survival by Hyperthermia. Photochem. Photobiol 42:533-540, 1985. 4) P.C. LEVENDAG et al.: Interaction of Interstitial Photodynamic Therapy and Interstitial Hyperthermia in a Rat Rabdomiosarcoma. Abstracts in Biological effect of Nonionizing Electromagnetic Radiation, 37-38, 1989 5) P. BUGELSKY, C.W. PORTER, T.J. DOUGHERTY: Autoradiographic Distribution of Hematoporphyrin Derivative in Normal and Tumor Tissue of The Mouse. Cancer. Res 41:4606-4612, 1982 6) J. DENEKAMP: Endothelial Cell Proliferation as a Novel Br. J. Cancer Approch to Targeting Tumor Therapy. 45:136-139, 1982 7) P VAUPEL: Hypoxia Res. 13: 399-408, 1977

in

Neoplastic Tissue. Microvasc.

8) J.T. LEITH, R.C. MILLER, E.W. GERNER, M.L. BOONE: Hyperthermic Potentiation: Biological Aspects and Applications to Radiation Therapy. Cancer 39:766s-779s, 1977 F.W. HETZEL, P. VAUPEL, T.S. SANDHU: 9) H.I.BICHER, Modifications by Localized Microwave Microcirculation Hyperthermia and Hematophorphyrin Phototherapy. Biblioth. Anat. 20:628-632, 1981 10) P.VAUPEL, S. FRINAK, H.I.BICHER: Heterigenous Oxygen Partial Pressure and ph Distribution in CH3 Mouse Mammary Carcinoma. Cancer. Res. 41:2008-2013, 1981 11) D.R. DOIRON, C.J. GOMER: Porphyrin Locaslization and Treatment of Tumors, Alan R. Liss Ed. 1984 12)

518

N.

AI-LAN, P. OIONG-OIAN: Studies on Hematophorphyrin-

photosensitized Effect on Human Cancer Cells in Vitro: TEM and SEM observations. In Methods in Phorphyrin Photosensitization, edited by D. Kessel, Plenum Press, 117122, 1985 13) D. KESSEL: Effect of Photoactivated Porphyrin at The Cell Surface of Leukemia Cells. Biochemistry L1210 16:3443-3449, 1977 14) I. FREITAS: Photodynamic Therapy and Hyperthermia:Common and Complementary Effects. Medecine Biologie Enviornment 14:93-111, 1986 15) T. CHRISTENSEN, J.MOAN, E. WIBE.R. OFTEBRO:Photodynamic Effect of Haematoporphyrin Thoughout The Cell Cycle of The Human Cell Line NHIK 3025 Cultivated in VItro. Br. J. Cancer 39:64-68,1979 16) J.OVERGAARD: Ultrastructure of Murine Mammary Carcinoma Exposed to Hyperthermia in Vivo. Cancer Res. 36:983.985,1976 17) P.S. LIN, F.H. WALLACH, S. TSAI: Temperature Induced Variations in The Surface Topology of Cultured Lymphocytes Are Revealed by Scanning Electron Microscopy. Proc. Nat. Acad. Sci. USA 70:2492-2496,1973 18) J.R. LEPOCK, P. MASSICOTE-NOLAN, G.S. RULE,J. KRUV:Lack of COrrelation Bewteen Hyperthermic Cell Killing, Thermotolerance, and Membrane Lipid Fluidity. Radiat. Res. 87:300-313,1981 19) D.B. LEPE:Molecular and Cellular Mechanisms of Hyperthermia Alone or Combined with Other Modalities. In: Hyperthermic Oncology, 1984, edited by J. Overgaard, Taylor and Francis, London, pp 9-40,1984 20) R.L. ANDERSON,G.M. HAHN:Differential Effects of Hyperthermia on the Na, K ATPase of Chinses Hamster Ovary Cells. Radiat. Res. 102:314-323,1985 21) G.M. BARRATT,E.D. WILLS: The Effect of Hyperthermia and Radiation on Lysosomal Enzyme Activity of Mouse Mammary Tumors. Eur. J. Cancer 15:243-250, 1979 22) C. TURANO,A. FERRARO,R. STROM,R. CAVALIERE, A. ROSSI FANELLI: The Biochemical Mechanism of Selective Heat Sensitivity of Cancer Cells III. Studies on Lysosomes. Eur. J. Cancer 6:67-72,1970 23) R. CAVALIERE, E.C. CICCATTO,B.C. GIOVANELLA, C, HEIDELBERGER,R.O. JOHNSON, M. MARGOTTINI, B. MONDOVI, G. MORICA, A. ROSSI FANELLI:Selective Heat Sensitivity of Cancer Cells. Biochemical and Clinical Studies. Cancer 20: 1351-1381,1967 24) K.G. HOFER, B. BRIZZARD,M.G. HOFER:Effect of Lysosomal Modification on the Heat Potentiation of Radiation Damage and Direct Heat Death of BP-8 Sarcom Cella. Eur. J. Cancer 15:1449-1457,1979

519

25) S.K. CALDERWOOD,E.A. BUMP, M.A. STEVENSON, R. GONZALESMENDEZ, E. SHUI, E. VAN KERSEN, G.M. HAHAN:INvestigation of Adenylate Energy Charge, Phosphorylation Potential, and ATP Concentration in Cells Stressed With Starvation and Heat. J. Cell. Physiol. 124:261-268, 1985 26) S.H. KIM, J.H. KIM, E.W. HAHN, N.A. ENSIGN: Selective Killing of GLucose and Oxygen Deprived HeLa Cells by Hyperthermia. Cancer Res. 40: 3459-3462, 1980 C.S. CHANG, M. TALLEN, W. BAYER, S. 27) P.N. YI, BALL:Hyperthermia induced Intracellular Changes in Tumor Cells. Radia. Res. 93: 534-544, 1983 28) R.A. COSS, W. C. DEWEY, J.R. BAMBURG: Effect of Hyperthermia on Dividing Chinese Hamster Ovary Cells and on Microtubules in Vitro. Cancer Res. 42: 1059-1071,1982

520