Dye-Sensitized Photoinactivation of Tumor Cells in Vitro
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Dye-sensitized Photoinactivation of Tumor Cells in Vitro JUmTH S. BELLIN,* STEVEN C. MOHOS, A~D GERALD OSTER (Department of Pathology, State University of New York, Do~zmslale Medical Center, and Department of Chemistry, Polytechnic Institute of Brooklyn, Brooklyn, N.Y.) SUMMARY The in vitro photodynamic inactivation of a wide variety of tumor cells was studied with various dyes used as sensitizcrs. The capacity of dyes to act as photosensitizers in this system parallels their capacity to sensitize the photoinactivation of many other substrates and correlates with their capacity to undergo photoreduction. Some dyes are able to inactivate tumor cells in the abscnce of light. :Photodynamic inactivation of the tumor cells dcstroys their tumor-producing capacity but docs not change their dyc- binding capacity. It has long been known that some microorgan- Menke (16) has shown that both normal and neo- isms, when stained with certain dyes, can be in- plastic cells grown in tissue culture (lie when sub- activated with visible light. This process, com- jected to the action of visible light in the presence monly referred to as photodynamic action, in- of a xanthene dye. The criterion of death in this volves the participation of oxygen and consists of author's publication was a subjective one, and, a dye-sensitized photoautoxidation of some es- since relevant controls were omitted, his results sential elements of the substrate involved. Many could be interpreted as being due to indirect ef- diverse substrates are affected by photodynamic fects, resulting from the photodynamic destruc- action. These include enzymes (~3), transfornfing tion of nutrients in the culture medium. principle (1), viruses (11), bacteria (10), and The present paper is concerned with the in vitro erythrocytes (8). The autoxidation of many simple photodynamic inactivation of tumor cells as organic substrates is enormously accelerated by judged by their inability to elicit tumor growth. light in the presence of certain sensitizing dyes. A In conformity with previous results on the photo- mechanism for the photosensitized autoxidation dynamic inactivation of transforming principle of para-toluenediamine and criteria for the capac- and para-toluenediamine, we have found that ity of dyes to act as sensitizers in such a system only certain classes of dyes can act as photo- were established (18). It was found that those dyes sensitizers in this system. which can photosensitize the autoxidation of aromatic amines can also act as sensitizers for the MATERIALS AND METHODS photodynamic inactivation of transforming prin- Tv,~ons A:ND DYES E.M1)LOYED ciple (1). The tumors used were: the Gardner lymphoma There is an analogous relationship between (6C3HED) grown in ~0-gram C3H/Hej mice (both photodynamie action and the effect of x-rays in the tumor and mice were obtained from the Jack- that both phenomena involve autoxidations. For son Memorial Laboratory); Sarcoma 180 and example, ferrous sulfate is autoxidized by the Ehrlich ascites tumor, both obtained from Dr. H. action of x-rays as well as by dyes and visible T. Sugiura at the Sloan-Kettering Institute and light; viruses can be inactivated by the action of both grown in ~0-gram Swiss mice from the both agents when oxTgen is present. Since some pathogen-free colony at the State University of tumor cells are susceptible to the action of ioniz- New York Downstate Medical Center; mammary ing radiation when oxygen is present, they might adenocarcinoma, dbrB, grown in DBA/1 mice, likewise be inactivated by photodynamic action. both obtained from the Jackson Laboratory. * Public Health Service Research Fellow of tile National Dyes (histological grade) were obtained from Cancer Institute. Eastman Kodak Company. The dye concentra- Received for publication April 10, 1961. tions employed were of the order of 10-5 M, so that 1365 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1961 American Association for Cancer Research. 1866 Cancer Research Vol. ~1, November 1961 the small amounts of added salts which these com- containing 100 units penicillin/ml, as detailed mercial samples contained should not influence above. the results obtained. DETERMINATION OF CELL VIABILITY: PROCEDURES FOR PHOTODYNAMIC TUMOR-PRODUCING CAPACITY INACTIVATION The samples of 6C3HED and dbrB tumor cells In the case of solid mouse tumors (6C3HED and were tested for their tumor-producing capacity by dbrB).--Growths were removed with aseptic pre- subcutaneous injection, 0.5 cc. of each suspension cautions, and a suspension of tumor cells was pre- being injected into each of four mice in the right pared by pressing the tumor through a 40-mesh or left axilla or groin. The animals were examined Monel metal sieve into physiological saline ad- daily for signs of tumor growth, and the earliest justed to pH 7.4. The suspension was diluted day such growth became palpable was noted. The with the same medium to give suspensions con- increase in tumor size as measured by palpation taining 106 cells per ml. as determined by hemo- was charted until the mice died. On autopsy tu- cytometer counts. All cell suspensions were mor-bearing mice showed huge localized tumor routinely tested for bacterial contamination by masses at the sites of injection. Previous experi- culture in trypticase soy broth at 37 ~ C. Aliquots ments in our laboratory have shown by methods of the counted suspension were pipetted into four of serial dilution that, under the experimental sterile test tubes, to two of which were added ali- conditions outlined above, Gardner lymphoma quots of a ]0-ZM sterile stock solution in saline of cannot be successfully transplanted if fewer than the dye under study. The final dye concentration 100 viable cells are injected. Since we implanted was 10 -~ M, except in those experiments in which it 500,000 cells, the fact that in "inactivated" was desired to investigate the effect of various samples no tumor growth was noted in more than dye concentrations. A dye concentration of 80 days means that in such samples fewer than 10 -5 M was routinely employed, because this con- 0.0~ per cent of the cells could have survived centration gives an optical density of about one photodynamic action. The suspensions of Sarcoma for most dyes at their respective absorption 180 and of Ehrlich ascites tumor cells were tested maxima. Unit optical density gives nearly optimal by the intraperitoneal injection of 0.5 cc. (500,- photometric efficiency. Of the resulting cell sus- 000 cells) into each of four mice. The first signs of pensions two (with and without dye) were kept in tumor growth, determined by the appearance of the dark at room temperature as controls, and ascites, were usually noted in 4-5 days, and death two with and without dye were illuminated in test followed about 10 days later. tubes at a distance of 15 cm. from the front sur- face of a 500-watt TDC brand slide projector. 1 BINDING OF DYES TO TUMOR CELLS This projector has a built-in heat filter and was Fresh suspensions of 6C3HED cells, obtained equipped with a Corning Number 3070 filter to as noted above, were washed with pH 7.4 saline eliminate radiation below 400 mtt. There was no and resuspended to a concentration of 107 cells/ml detectable rise in temperature of the illuminated of suspension; 0.0~ ml. of 10-3M dyes were added samples. The samples were illuminated for 30 to duplicate ~-ml. aliquots of this suspension. minutes, except where otherwise noted. One sample was illuminated for 30 minutes, and The suspensions were then centrifuged in a one was kept as a dark control at room tempera- size 1, model CM International Centrifuge, with ture. Two other samples of the suspension, to a #s head, at 800 r.p.m, for 10 minutes, after which no dye had been added, served as illumi- which the ~sediments were resuspended in their nated and dark controls. After illumination all original volume of sterile saline containing 100 samples were centrifuged at 800 r.p.m, for 10 units penicillin/ml. minutes; the resulting supernatants (which were In the case of ascites tumors (Sarcoma 180, Ehr- lich ascites).--Tumor cell suspensions were har- not turbid) are referred to as S1. The sedimented vested 5 days after inoculation as milky-white sus- cells were washed with pH 7.4 saline, and the re- pensions, which were tested as above for bacterial sulting supernatant was labeled S~. By means of contamination. To diluted suspensions, containing a Zeiss spectrophotometer, the optical density at 106 cells per ml. of pH 7.4 saline, dye was added to the absorption maximum of each dye in question a concentration of 10 -5 M, and aliquots were illumi- was then determined in (a) the solution containing nated, centrifuged, and resuspended in saline only 10 -5 M dye in saline, (b) supernatant S1, and (c) supernatant S~. From the difference in optical 1 Manufactured by Bell and Howell Company, Chicago, Illinois. density of the appropriate solutions and the molar Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1961 American Association for Cancer Research. BELLIN et al.--Photoinactivation of Tumor Cells 1367 extinction coefficient of the dye in question, the experiments are summarized in Table 3. With amount of dye bound to the cells was determined. 10 -5 M methylene blue or proflavine, 5 minutes' illumination gave only partial inhibition (100 per RESULTS cent takes, but after an increased latent period); Photodynamic inactivation of tumor ceUs.--In with 10 minutes of illumination at the same dye Table 1 are summarized experiments demonstrat- concentration complete tumor inhibition was ob- ing the photodynamic effect on 6C3HED tumor served. Rose bengal, as previously noted, had a cells of thirteen dyes which were chosen as repre- "dark effect," and was able to inactivate the tumor sentative members of the various families of dyes; cells even in the absence of light.