Haim I. Bicher and D. F.

Bruley

Western Tumor Medical Group, Valley Cancer Institute 5522 Sepulveda - Van Nuys, Ca. 91411 - - Louisiana Tech University - Bicmedical Engineering Department - Ruston Louisiana 71272

Due, in part, to increasing clinical interest, active investigation of the physiological phenunena induced by hyper~a is in progress (1, 2, .7, 13). The studies of Eddy (7) and Reinhold (13) employing "ch~r systzns" have both shown changes in the microvascular network as a function of temperature an~ exposure ti,~. Cater et al. (3) reported on changes in tmor oxygen tension with hyperthermia but did not record changes in tumor t~m.rature. Bicher (I, 2), in a mouse leg t~or system, reported that tmor blood flow increased up to 41C and then decreased to 44oc. The oxygen tension in the truer, as measured with a platinum electrode, generally followed the changes in tu~or blood flow. Althcugh blood flow and shifts induced in It by hy~a in both tmor and normal tissue is important, several other parameters also "have significant roles. Several studies indicate that the ~ of interstitial fluid in h~an and rex:lent solid, tumors is .3 to .5 units lower than the normal tissue p~ of about 7.4 (8, I0). P~o~ced ;S has also been shown to affect the transpL%ntability of tt~or cells heated in vitro (12). In a recent paper, ~ has shown (9] that there is a variable influence of according to tmperatu~eand that thare is a oritical point in the increased lethality of heat below pS 6.7. -.*This work was partially reported at the llth European Conference for Microcirculation, Garnish, 1981 (Bibl.Anat. N-20). Thanks are given to Mr. S. Frinak for technical assistance in part of this work. 623

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H. I. BICHER AND D. F. BRULEY

In a~diticn to hyperthermis, He~atoporphyrin derivative (HpD)

phototherapy is aL~o showing so~e pronise in clinical cancer theraI~

(5, 6). This type of therapy employs an injectable dye (HpD) which is specifically aco~ulated in so~e tuners and/or is specifically cleared fro~ normal tissues (5, 6). When light of specific wavelengths illuminate the dye a photochemical reaction takes place yielding the cytmtoxic agent singlet oxygen (7, 15). Although the exact ~echanim~(s) of tunor inactivation have yet to be detezmined, the rapid and dramatic coloration changes observed in tumors following treatment s~ggest that modification of blood flow with possible conconitant effects on pO2 and p~ may play a pruninant role (5).
MATSR.T.ALS AND F~TH(X)S

Animal syst~. All in situ studies were carried out in 4th, generation transplants of C3H ma, rnary adenocarcincma implanted in t~ hind leg of C3H SED-BH mice. This is a syngenetic implantable tumor that is kept at o~r facility using solid tissue transplants that are inoculated subcutaneously into recipient mice. T~ors used for experimentation were approximately 8-1G~m in diameter. The mice were anesthetized during microelectrode introduction with a combination of Ketmnine 40mg/kg I.M. and Thorazine, 5~g/kg I.M.

Oxygen ultramicroelectro~es. The 02 ultramicroelectrodes used were as described by Cater and colleagues (4). They were made by pulling a glass tube (KG-33 , ]D l.Snm, ~D 2.0m~, Garner Glass Co., Clar~mont, California), encasing a 20- ~ gold wire (Sigmund Cohn Corp., Mr. Vernon, New York) in a David Kopf Model ?00C vertical pipette puller. The exposed gold ti,~ is about I0, in diameter, and is coated with a Rhoplex (PJ~m Haas, Philadelphia, Pennsylvania) menbrane as previously described (2). This probe is used as an "external reference" ~ microelectrode. Electrodes are calibrated as previously described (I) in buffered saline solutions of known .P9.2 values. The electrodes are conditioned by placing ~J~n in buffered saline and applying 0.8 V potential for 2 hrs. After this treatment they are usually very stable. The current reading at zero oxygen tension is very (residual current) and the response of the microelectrode to ~2mnges of oxygen tension is very rapid.,

In these experiments a polarizing voltage of 0.6 V has been used. The relationbetween current output an~ oxygen tension is linear, the current pernm Hgbeing of the order of magnit~e of 0.6 x 10-11A. ""
1~1 ultramicroelectrodes. Designs for glass pH microelectrodes employed in this st~J have been developed, m~st notably by Hinke

(ii). The Hinke-type electrode consist of a 1~ sensitive glass

CHANGES IN TISSUE OXYGENATION AND ACIDITY

625

micropipette inside of pyrex glass pipette with the tip of the ~ sensitive micropipette extruded from the pyrex glass pipette. A silver/silver chloride electrode is inserted into the electrode ste~ which is filled with 0.1 N HCI. This type of micz~e.lectro~e is an advantage ~ver other types of microelectro~es in. which a recessed tip may cause a response ~ of up to several minutes. T~perature d.eterminaticns. Tumor and meuse core t~.ratures were recorded using Copper-C~nstantan microthermmmm~les (tip diameter 30-100 microns - ~ERA Inc. ) inserted into the ~ral t.issue in close proximity to the microelectrode or in the animals rectum for core measurements. An Omega Engineering Model 250 Digital Voltmeter amplifier was used as a link between the microthermocouple and the polygraph. Microwaves of a frequency 2450 ~z were produced by a Paytbeon Magnetron and delivered through a specially designed 2cm square applicator loaded with ~w loss dielectric material having a dielectric constant of 6 {14). H~O phototherapy. Tumor-bearing ~ice were injec,ted with kg HpD and 24 hours later were exposed to the light Source. The mice were shielded from the light during the 55 minute exposure except for the tuner-bearing region. The light source was a mDdified Bessler lantern slide projector which was filtered .to yield 150~9/cm2 at the tumor surface over the rang. e 600-730nm.

4 different major e.xperin~nts were performed. In the first pO2 distribution after 1 hour hyperthermia was studied l, 4 an~ 24 hours after trea~.nt. The second experiment was similar but studied the effect of Phototherapy on TpO~, at_ similar ti~.e intervals. Experiment 3 studied the effect of hypertSermia cn tissue at similar time intervals, while experiment 4 determin~ an~ the effect of Hyperthermia and phototherapy on tissue pH. All results are expressed in Histogram fashion.
Experiment I: Effect of hyperthermia on T~ - time ~orrelati~. Histograms were obtained before and l, 4 and 24 /~urs after microwave induced hyperthermia (42-43oc) for ode hour. These results are sho~ in Figure i. There is a progressive shift towards I~ ..pO2 values, with all the tu~or virtually hyp~xic after 24 h~urs. No reoxygenation is noted. Experiment 2: Effect of Phototherapy on T~O2 - Time correlation. Histograms were obtained before and i, 4 and 24 hours after H~ phototherapy (20,g/kg Hpd, light exposure 150 ~9/c, for 55 minutes). The p~ histogram shifts tm~ard the hyp~xic region (0.5 m~ Hg) quickly. Huwever, at 4 hours there is an attempt to
reoxygenate, as sb:~n by the reappearance of an Oxic tail in the

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H. I. BICHER AND D. F. BRULEY

VALU[S |[fOl~ AND

NyP[IITI4~ItMkt

Fibre 2. Effect of Hid Phot;otbe-~al:b ~ TL~O2

CHANGES IN TISSUE OXYGENATION AND ACIDITY

627

20-40 nm Hg region. At 24 bour.~ all values are between 0-10 ~hese results are shown in Figure 2.

Experiment 3: E~fect of Syperthermia on TpH. Time correlation. Hist,!rams obtained before and i, 4 and 24 hours after microwave reduced H~a (42-43C) for one hour. Results shoan in Figure 3. Note a remarkable shift of the tissue pH values towards acidity ~ith mean TpH changing frcm 6.74 before heat to 6.21 after. TpH r~ins in that region at 4 and 24 hours.
Experiment 4: Effect of Hypartharmia and Phototherapy ~n Tissue p~ - a correlation. Histograms obtained before or i hour after microwave induced Byperthermia, Pbotmtherapy or a cu%bination of both. Note that the pH shift tuwards acidosis (same as sh~ in figure 3) induced by hyperthermia is not present after Phototherapy, This correlation is shown in Figure 4.

Determination of the mode of tuner inactivation by a treatment m~ality is critical to its develop,ent and future use. It has been shmn that hyperthermia has many possible effects on cell survival either alone or in combination with radiation (9, 12, 13). It has also been r~ho~ to dramatically modify bloc~ flc~ and oxygenation within tu~ors (i, 2). The results presentad ~ere indicate that a significant reduction in pH is induced by hyperthermia %~dch may result in a significant increase in cell killing within the tar (9). It is possible that *h~s observed pH r~ift is due to a cc~binatic~ of hyperthezmia stinulated cellular metabolic activity and the simultaneous reduction in tu~or blood flow which is observed at the treatment temperature (i). The effects seen on pH end pO~ following H~O photott~apy are quite different fzn~ those follo~i~g hyperthermia. It is clear that there is a sharp reduction in pO2 at all areas within the tm~r without any significant shift in pH. Although further studies are now in pzDgress, it is possible to speculate on the meardng of the results presented here. Massive coagulation necrosis within tumors is reported to foll~ HpD phot~therapy (5).. It is likely that the cells ~ost affected by this treatment are the vascular endothelial cells o the tmor microvasculature. Their destruction ~uld result in the observed coagulation necrosis with an abrupt reduction in blood flow. This ~ould result in the 1oa leveAs of tissue oxygenation reported here. Since there is no ~llular metabolic stimulation with this modality and there is direct cyto-

toxicity (15) no dramatic shift in ~ would be ~ and n~ne

wae,observed.

Further s,mdies are currently in progress to further examine the effects of hyperthermia an~ H~D phototherapy ~ tmor

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H. I. BICHER AND D. F. BRULEY

F~.gure 3. Effect of Hypertherm.ia on T~.

C3H MOUSE MAMMARY CARCINOMA

140)

Figure 4. Effect of Pbot~tbe.ral:~,, Xyperthea~a or a ~mbination of both ~n TI~.

CHANGES IN TISSUE OXYGENATION AND ACIDITY

HICROPHY$1OLOGY

629

4. PhototherapT:

A) Ktcroc~rculit|on Di~age. B) 0z Histogram shifts toward 0-$ mm Hg qu|ckly. C) Reoxygenition at 4 hours. D) Ko pH

Figure 5.

For explanation see text.

microphysiology. ~esults obtained so far a.re su,~arized in Figure 5.

Changes in t~r tissue oxygenation and acidity were determined

using ultramicroelectrodes, and presented in his.togr~n fashion.

The effect of H~a a~d HF~ p~o~o-therapy were teste~. It was our~ that both mcdalities affect t~or microcirculatton, causing a marked drop in ox~en availability. Tissue p~ is decreased by Hy~ertharmia, ~ut not by phototherap7. These effects

are long lasting, at least for 24 hours after treat~nent.

I. Bicher, H. I., Hetzel, F. W., Sandhu, T. S., Frinak, S., Vau~el,

P., OHara, M. D. and OBrien, T. Effects of hypertb~zmia

on normal and tu~)r micrcenviron~ent. Radiology, In Press 1980. 2. Bicher, H. I. Increase in brain tissue oxygen availability induced by localized micro~ve hyperthez~a. In Silver, Erecinska, and Bcher, Oxygen transport to tissue; Vol. IZI, ~. 3~7-353, Plen~ Press, Ne~ York, 1978. 3. Cater, D. B. and Silver, I. A. @~antitative measure~nts of oxTgen tension in normal tissues and in the t~m:rs of patients before and after radiotheral~. Acta. Radiologica. 5.~3: 233-256, 1960.

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H. I. BICHER AND D.~F; BRULEY

4. Cater, D. B., Silver, I. S. and Wilson, G. M. Apparatus and technique for the quantitative measurement of oxygen tension in living tissues. Proc. Roy. Soc., lend. (Serie~ B) 151: 256-276, 1959-1960. 5. Dia,ond, I., McDonagh, A. Fi, Wilson,~ C. B., Granelli, S. G., Nielsen, S. and Jaenicke, R. Photodynamic .therapy of malignant t~rs. Lancet, 2: ii.75-I177, 1972. 6. Do~herty, T. J., Kaufman, J. E., Goldfarb, A., Weishaupf, K. R~, Boyle, D., and Mittle~an, A. Photoradiation therapy for the treatment of malignant tumors. Cancer Res. 38: 2628-2633, 1978. 7. Eddy, H. A. Alterations in tumor microvasculature dttring hyperthermia~ Radiology, In Press, 1980. 8. Eden, M., and Kahler, H. The pH of rat t~rs measured in vivo. J. Natl. Cancer Inst. 16: 541-556, 1955. 9. Ger~t=ck, L. E. Modification of cell lethality at elevated temperatures: The pH effect. Radiat. Res. 70:224-235 1977. i0. (Atllino, P. M., Grantham, F. M. and Smith, S. H. Modifications of the acid-base status of the internal milieu of tumors. j. Natl. Cancer Inst. 34: 857-860, 1965. ii. Hinke, J. A. Cation-selectivemicroelectrcdes for intracellular use. In Eiserman, Glass electrodes for hydrogen and other cations; Marcel Dekker, Inc., New York, 1973. 12. Overgaard, J. Influence of extracellular pH on the viability and morphology of tumor cells exposed to hyperthermia. J. Natl. Cancer Inst. 56: 1243-1250, 1976. 13. Reinhold, H. S., Blachiewicz, B., and Berg-Blok, A. V. D. Decrease intumormicrocirculationduring h.yper~a. in Streffer, Cancer therapy byhypert~a and radiation. pp. 231-232, Urban and Scbwarzenburg, Baltimore, 1978. 14..Sand_dhu, T. S., Kowal, H. S., and Jo~du~on, R. Development of hyper~a applicators. Int. J. Radiat. Oncol. Biol. Phys. ~: 515-519, 1978. 15. Weishaupt, K. R., Gcmer, C. J. and Do~gherty, T. J. Identification of singlet oxygen as the cytotoxic agent in photo.inactivation of a~urine tu~or. Cancer Res. 3--6, 2326, 1976.