MECHANISMS INVOLVED IN THE PRODUCTION OF RED

FLUORESCENCE OF HUMAN AND EXPERIMENTAL

TUMOURS

F. N. GHADIALLY,
W. J. P. NEISH AND H. C. DAWKINS
Department of Pathology, Cancer Research Unit and Department of
Bacteriology, University of Shefleld

ULCERATED squamous carcinomata of man show a red fluorescence

when examined in ultraviolet light (Gougerot and Patte, 1939;
Ronchese et al., 1954; Belisario, 1959); Ghadially (1960) found that
carcinogen-induced squamous carcinomas in the rabbit, mouse,
hamster and hedgehog exhibit a similar fluoresence. It has been shown
(Ghadially and Neish, 1961) that the red fluoresence of rabbit carcinoma
is due to the presence of protoporphyrin and also perhaps to traces of
coproporphyrin.
Little, however, is known about the factors involved in the pro-
duction of this red fluoresence. Two main possibilities have to be
considered: (1) that the porphyrins are the product of microbial
activity on the ulcerated surface of the tumour, and (2) that the
porphyrins are of host origin and are excreted from the circulating
blood by the tumour. This paper reports experiments carried out to
test these two theories.
We have found that some common organisms such as staphylo-
cocci and coliform bacilli isolated from the surface of squamous
carcinomas can produce red fluorescence when injected into a number
of transplantable mouse and hamster tumours. Since these organisms
do not produce red fluorescent colonies on common laboratory
culture media it seemed to us that the turnour or its host was providing
some material lacking in the culture media employed. Arguing along
these lines we conducted a series of experiments, which finally led to
the formulation of a new culture medium containing delta-amino-
laevulinic acid (DAL). When organisms capable of producing red
fluoresence in tumours are grown on this medium brilliant red-
fluorescent colonies are readily obtained.
Another series of experiments was carried out in which proto-
porphyrin was injected into tumour-bearing animals. This showed
that circulating porphyrins can readily accumulate in tumour tissue
and that they can also appear on the surface of ulcerated squamous
arcinoma.
I . PATIS. .ACT.-VOL-. 85 (1963) 77
78 F. N. GHADIALLY, W. J. P. NEISH AND H. C. DAWKINS

MAT~RIALS AND METHODS

Tumours
Crocker carcinoma. This tumour has been carried by us in serial transplanta-
tions for many generations in commercially obtained albino mice. It is trans-
planted by inoculating subcutaneously in the right flank 0.2 ml. of a thick pasty
su'spension of tumour mince, containing 100 mg. streptomycin base and 50,OOO
Units crystalline penicillin per ml. The whole procedure was carried out with
strict aseptic technique.
Transplantable squamous carcinoma of hamster. This turnour, which arose in
the skin of a brown hamster painted with 9:lOdimethyl-l:2-benzanthracene
(DMBA) (Illman and Ghadially, 1960), has now been carried in serial transplants
in brown hamsters by subcutaneous inoculation of tumour paste with added
streptomycin and penicillin (v. supra). It is a slow-growing cystic tumour with a
take rate of approximately 60 per cent.
Rabbit squamous carcinoma. These turnours were produced by painting
rabbits once a week with a 2 per cent. solution of DMBA in a mixture of equal
parts of lanolin and paraffin (Ghadially, 1958). Three lots of six rabbits were
placed on this regime at four-month intervals so as to provide a supply of carcinomas
and red-fluorescent material throughout the course of the experiment.
Hyjmmelanotic melanoma. This turnour arose in the skin of a white hamster
painted with DMBA (Illman and Ghadially, 1960). It has now been carried by
serial transplants in white and brown hamsters with the aid of streptomycin and
penicillin. It is a slow-growing tumour which only occasionally produces a little
melanin.
DAL blood agar
This medium on which certain bacteria produce red-fluorescent colonies was
made up as follows: a solution containing 5 mg. delta-amino-laevulinic acid
methyl ester hydrochloride (L.Light and Co. Ltd. Colnbrook, England) per ml.
in distilled water was prepared; 1 ml. of this was added to 9 ml. of warm melted
7 per cent. blood agar. This was poured into a petri dish and allowed to set in
the usual manner. The plates were dried in the incubator before inoculation with
organisms.
Bacteria
Rabbit pasteurella. This was isolated from the surface of an ulcerated red-
fluorescent squamous carcinoma produced on the ear of a rabbit by DMBA; it
produces red fluorescence when injected into Crocker carcinoma.
Staphylococcus pyogenes. This was isolated from the surface of an ulcerated
squamous carcinoma of man; it also produces red fluorescence when injected
into transplanted animal turnours.
Other organisms. Many other organisms-ither stock cultures maintained
in our laboratory or obtained from the National Type Culture Collection-have
been grown on DAL blood agar to test their power to produce red fluorescence
in this medium.
Ulcers
From the flanks of rabbits and mice sufficient skin was excised with scissors
to produce an ulcer approximately 3 cm.diameter in the rabbit and 1 cm.diameter
in the mouse. Ulcers produced 4 days before the animal was used in an experiment
will henceforth be referred to as old ulcers, those produced 24 hr before the test
will be referred to as recent ulcers.
Protoporphyrin solution
This was made by dissolving 200 mg. of protoporphyrin IX (L. Light and Co.
Ltd) in a solution of 200 mg. of NaZCO3 in 50 ml. of distilled water.
 RED FLUORESCENCE IN TUMOURS 79

H u o r e ~ ~ nphotography
ce
The light source was the Leitz fluorescence microscopy unit (Messrs E. Lei@
London). Photographs were taken with a Leica M3 fitted with bellows focusing
unit, reflex housing and anf4-5lens of 13-5 cm.focal length (Hector). Fluorescence
was recorded on 35 mm. Daylight type Ektachrome with an ultraviolet filter
(Wratten 2A) in front of the camera lens. The length of exposure required varied
from 4 to 5 seconds at f4.S-5.6.

EXPERIMENTAL
Experiment 1
Approximately 5 mg. of necrotic red-fluorescent material scraped
off the surface of an ulcerated squamous-cell carcinoma of the rabbit
was shaken with 50 ml. of sterile saline. The suspension was allowed
to stand for a few minutes, and the resultant supernatant fluid was
decanted and divided into two portions, one of which was sterilised
by boiling for 15 min.
Twelve mice bearing Crocker carcinoma transplants c. 2.5 cm.
in diameter were selected for the experiment. The tumours of 6
were injected with 0.2 ml. of the suspension described above (experi-
mental group), and the tumours of the other 6 mice were injected
with the boiled suspension (control group).
One week later all the surviving mice with intact non-ulcerated
tumours were killed, and their tumours were sliced and examined
under ultraviolet light. The tumours of 4 experimental animals showed
areas of red fluorescence, but none of the tumours in 5 control animals
showed it. The areas of red fluorescence corresponded with the areas
of necrosis in these tumours. It was therefore decided to see whether
red fluorescence could be produced as a result of simple chemical
necrosis.
Experiment 2
Forty mice bearing Crocker carcinomas c. 2.5 cm.diameter were
divided into four equal groups. Tumours of group 1 mice were
injected with 0.2 ml. of 0-1N-NaOH, of group 2 with 0.2 ml. 0 . 1 ~ -
HCI, of group 3 with 0.2 ml. of absolute alcohol and of group 4 with
0.2 ml. of turpentine. At the end of a week there were 6 survivors in
group 1, 4 in group 2, 3 in group 3 and 3 in group 4. There was
abundant necrosis in all these tumours, but none of them showed the
characteristic red fluorescence. The result of this experiment taken
in conjunction with that of experiment 1 indicates that tumour necrosis
alone is not sufficient for the development of red fluorescence, and
that one or more organisms destroyed by boiling may be involved.
Experiment 3
An attempt was now made to isolate the bacteria responsible for
the red fluorescence. Material from the surface of a red-fluorescent
rabbit carcinoma was plated out on blood agar and incubated at
37" C. for 24 hr. From this plate three organisms were isolated and
80 F. N. GHADIALLY, W. J. P. NEISH AND H. C. DAWKINS

subcultured in nutrient broth : a coagulase-positive staphylococcus,

a coliform bacillus and a pasteurella.
Fifteen Crocker-carcinoma-bearing mice were divided into three
equal groups and their tumours injected with 0.2 ml. of culture of
each of these organisms. After a week all surviving mice were killed.
The tumours of 2 survivors receiving the coagulase-positive staphylo-
coccus showed a pale pink fluorescence. The 2 survivors receiving
coliform bacilli showed no red fluorescence, but all 3 survivors receiving
pasteurella showed red fluorescence in their tumours (figs. 1 and 2).
Thus it would appear that the red fluorescence in the Crocker
carcinoma was produced mainly by the pasteurella and it was decided
to investigate whether the organism or its products was responsible
for this phenomenon.
Experiment 4
Twenty-seven albino mice bearing Crocker carcinoma transplants
c. 2-5 cm. in diameter were divided into three equal groups. Each
tumour in the first group received one injection of 0-2 rnl. of a 36-hr
TABLEI
Fluorescence of Crocker carcinoma given injections of pasteurella culture,
Seitz filtrate of the culture, or Hartley's broth

Proportion of tumours showing red fluorescence

Time after after they had received injections of
injection
(days) I! rabbit pasteurella -
. Seitz filtrate Hartley's broth
I I

... -.-
...
, i
...
018 1
!
oji

broth culture of rabbit pasteurella. The second group received a

Seitz filtrate of the same culture, and the tumours in the third group
were injected with the same amount of Hartley's broth. In previous
experiments an arbitrary period of 7 days was allowed to elapse before
the potency of an agent to produce fluorescence was assessed. In
this experiment, however, the tumours of mice that died during the
course of the experiment were examined as well as those of mice killed
at the end of the experiment at 10 days. An ulcerated tumour was
discarded from the experiment. Table I shows the results obtained.
It will be observed that only the actual culture of pasteurella produced
red fluorescence in the Crocker carcinoma.

Experiment 5
This experiment was designed to test the power of some stock
organisms to produce red fluorescence when injected into the Crocker
 NEIWAND DAWKINS
GHADIALLY. PLATEXI

RED FLUORESCENCE IN TUMOURS

FIG.1 .<rocker carcinoma of the FIG.2.P"me tumour as in fig. 1

mouse injected with pasteurella viewed under ultraviolet radia-
isolated from rabbit carcinoma. tion. The necrotic areas show
a strong red fluorescence.

FIG.3.-Mouse muscle FIG.4.P"melesion as in

injected with pasteu- fig. 3, to show pale red
rella isolated from fluorescence under ultra-
rabbit carcinoma. violet radiation.

FIG.5.-Divided plate containing FIG. 6.--Same plate as in fig. 5

blood agar in the right half and
DALblood agar in the left half.
Both sides seeded with pasteu-
rella isolated from rabbit
carcinoma.
viewed under ultraviolet radia-
tion showing red fluorescent
colonies produced on DAL-
blood agar.
 RED FLUORESCENCE IN TUMOURS 81

carcinoma. The organisms tested were: Staphylococcus pyogenes

(Oxford H strain), Escherichia coli, Cand& albicans, Streptococcus
pyogenes (group A), Pastewella septica (man), and Pastewella septica
(mouse).
Tumours of mice bearing 2.5 cm. Crocker carcinomas received
injections of 0-2 ml. of a Whr broth culture of each test organism.
Two controls were included in each test: a negative control of mice
with uninjected tumours to show that the tumours had not become
accidentally contaminated with an organism capable of producing
red fluorescence, and a positive control consisting of tumours injected
with a culture of rabbit pasteurella known to produce it. Each test
TABLE 11
I?!uorescence of Crocker carcinoma given injections
of various bacteria

Broth culture I noportion of tumours ~ntensityof red

injected 1 showing red fluorescence fluorescence
I I
. I
Staphylococcuspyogenes (Oxford strain)
Escherichia coli . . . . . 8: i
strong
moderateto
strong
Candidaalbicans . . . . .
Streptococcuspyogenes b o u p A)
Pastewella septka (from man)
.
.
.
.
Pastewella septicu (from mouse) . . I moderate

group consisted of 5-10 mice, with 3-4 negative and 3-4 positive
controls.
The intact tumours of all mice dying in 5-10 days or killed at
10 days were examined and the results recorded (table 11). The
controls behaved as expected. None of the 28 uninjected tumours
showed red fluorescence, whilst all 22 of those injected with rabbit
pasteurella showed it. It is obvious from this experiment that many
apparently unrelated species of organism can produce red fluorescence
when injected into the Crocker carcinoma.

Experiment 6
Two organisms (Staph.pyogenes and Escherichia coli) were isolated
from a swab taken from the ulcerated surface of a red-fluorescent
squamous-cell carcinoma of the leg of a woman aged 50 yr.
The tumours of 7 Crockerarcinoma-bearing mice received
injections of broth cultures of each of these organisms. After 8 days
all surviving mice were killed and their tumours examined for fluores-
cence. The tumours of 4 inoculated with Staph. pyogenes and 5 with
E. coli showed well marked red fluorescence. The strongest fluorescence
was seen in some of the tumours into which Staph. pyogenes had been
injected.
1. PATH. MIX.-VOL. (1963) P
82 I;: N. GHADIALLY, W. J. P. NEISH AND H. C. DAWKINS

Experiment 7
Six hamsters bearing transplantable squamous carcinomas were
divided into two equal groups. The fluid centre of the cystic
tumours in one group was aspirated and 0-2 ml. of a broth culture of
rabbit pasteurella was injected into the cystic cavity. The other
unaspirated, uninjected group served as controls. Fifteen days later
all the animals were killed and their tumours examined. The injected
tumours had been almost entirely reduced to a pultaceous mass showing
brilliant red ultraviolet fluorescence, far surpassing any that we had
hitherto witnessed.

Experiment 8
Six hamsters bearing hypomelanotic melanoma transplants (c.
1.2 cm.) in the wall of the cheek pouch were divided into two equal
groups. The tumours in the experimental group were injected with
0.1 ml. of a broth culture of rabbit pasteurella; those in the control
group were not injected. Fifteen days later the animals were killed
and their tumours examined. All the tumours in the experimental
group showed well marked red fluorescence whilst all the tumours in
the control group were negative.

Experiment 9
Though red fluorescence is usually seen on ulcerated human
squamous carcinomas it is also occasionally detectable on some non-
malignant chronic ulcers of man (Sharvill, 1955). It was therefore
TABLE I11
Fluorescence of mouse muscle that had received injections of
various bacterial and chemical agents

Material injected NO. of mice

injected
ii NO.. of
survivors
Proportion of survivors
rhowing red-fluorescent
muscle

Streptococcus pyogenes (group A) . 14

i
' 14 0114
Rabbit pasteurella . . . 18 1 15 6/15
Escherichia coli . . . . 14 j 12 9112
Staphylococcus pyogenes . 14
j 13
9/13
0.1 N-NaOH . . . 7 l 7 017
0.1 N-HCI . . . . 7 017
Turpentine . , . . 7 017

decided to see whether the organisms capable of producing red

fluorescence when injected into the Crocker carcinoma would also
produce red fluorescence when injected into the muscles of normal
mice. At the same time it was decided to observe whether or not
necrotic areas produced in muscle tissue by the injection of chemical
 RED FLUORESCENCE IN TVMOURS 83
agents would show red fluorescence. Groups of normal mice were
therefore given injections of 0.2 ml. of various cultures and chemical
agents into the muscle mass of the right thigh (table III). The surviving
animals were killed at 7 days and the muscles examined. Table I11
shows that chemical necrosis does not yield positive red fluorescence,
but various bacterial agents can do so (figs. 3 and 4). The organisms
capable of producing fluorescence are the same in muscle as in tumour
tissue. Thus pasteurella isolated from rabbit carcinoma, and Staph.
pyogenes and E. coli isolated from human carcinoma, can produce
fluorescence in Crocker carcinoma as well as in normal muscle, but
Str. pyogenes is incapable of producing fluorescence in either tissue.
The red fluorescence produced by various bacterial agents in muscle
was rarely as brilliant and never as extensive as that seen in tumours
nor could the phenomenon be reproduced so consistently (cf. tables I1
and III).
Experiment 10
The production of red fluorescence in the muscles of normal mice
by bacterial agents raised the question whether or not this phenomenon
could be reproduced in other animal species. Four rabbits, 4 rats
and 4 hamsters were given injections of a 24-hr culture of rabbit
pasteurella into the right thigh muscles, 0.5 ml. for each rabbit or rat,
and 0.2 ml. for each hamster; two days later all the animals received
a second injection of the same dose of pasteurella culture at the same
site. At 5 days one of the rabbits died and the muscles of its right
thigh exhibited large necrotic red-fluorescent areas. The right thigh
muscles of a rat and a hamster killed on the same day did not exhibit
any red fluorescence. At 7 days one animal of each species was killed;
only the rabbit showed a positive red fluorescence in its injected muscle.
All the remaining animals were killed at 12 days and again red
fluorescence was detected in the 2 rabbits, but not in 2 rats or 2
hamsters. It would appear therefore that there is a species difference
as far as fluorescence in normal tissue is concerned. Such a difference
is not, however, seen in turnours, for transplantable hamster tumours
can be made to show red fluorescence, as do ulcerated rat tumours
(Ghadially, 1960).
Experimeat 11
In view of the species difference observed in the previous experiment
and the uncertainty regarding the occasional red fluorescence believed
to occur in some human non-neoplastic ulcers it seemed worth while
to decide whether red fluorescence could be produced in the muscles
of a species more closely related to man than common laboratory
rodents.
A young male Macacus rhesus monkey received 1 ml. of a broth
culture of Staph. pyogenes by injection into the right thigh and 1 ml.
of a culture of E. coli into the left thigh. Both these organisms had
been isolated from a human carcinoma. Two days later the injections
84 F. N . GHADIALLY, W . J . P. NEISH AND H . C. DAWKINS

were repeated. At 7 days the animal was killed and its thigh muscles
were examined under ultraviolet radiation. Though many areas of
inflammation and necrosis were evident they did not show red
fluorescence.
Experiment 12
During the course of this work we discovered that the gastro-
intestinal contents of mice maintained on diet 86 showed a dull red
fluorescence. An examination of samples of the various constituents
of this diet under ultraviolet radiation revealed that only one of them,
grass meal, showed this type of fluorescence, which, as is well known,
chlorophyll can under certain circumstances show. We therefore
TABLE 1V

1 %;zf,
Effect of diet on redfluorescence

Bacteria injected 1 Diet

I No. of
survivors
Proportion of
survivors showing
red fluorescence
I I
I I I
.
Streptococcus pyogenes
(group A) 1 Brown
Diet
bread
86 6 ,
016
116
Pasteurella . . . Brown bread
Diet 86 6 i
6
6
6
616
516
Staphylococcus pyogenes .
1 Brown bread
Diet 86
I
I 1 5
5

decided to find out whether this diet was in some way responsible
for the red fluorescence seen in previous experiments.
Eighteen mice were given a diet of brown bread (which does not
fluoresce red under ultraviolet radiation) for 10 days and another
18 received diet 86. They were divided into groups of 6 animals each
and cultures of various organisms were injected into their right thighs.
Table 1V shows that the type of diet used did not materially affect
the results. Str. pyogenes, as in previous experiments, failed to
produce fluorescence in tumours and muscles alike; the single positive
observation in this group (table IV) might be attributed to accidental
exogenous or endogenous contamination of the tumour by some
other organism.
Experiment 13
The aim of this experiment was to determine whether red-fluorescent
colonies could be produced in vitro by growing organsisms on media
containing tumour tissue. Sterile slices of Crocker carcinoma were
set in petri dishes containing warm melted nutrient agar and warm
melted blood agar. Plates were also prepared by mixing approximately
one part of finely minced tumour tissue with two parts of nutrient
agar or blood agar.
 RED FLUORESCENCE IN TUMOURS 85

Two organisms capable of producing red fluorescence in trans-

planted tumours, pasteurella isolated from rabbit carcinoma and
Staph. pyogenes isolated from human carcinoma, and an organism
incapable of producing red fluorescence (Str. pyogenes) were used
as test organisms. These were seeded on the media described and
incubated for 4 days at 37" C. A duplicate set of plates was kept at
room temperature (20" C.). All plates were examined at 24-hrintervals
under ultraviolet radiation ; no red-fluorescent colonies were detected
in any instance.
Experiment 14
Since red fluorescence was not obtained by growing organisms on
tumour tissue in vitro it seemed to us that the missing factor lay not
in the tumour but in its host. This led to the idea that the essential
substance resided in the circulating blood, and that bacterial activity
on the surface of the tumour converted it into red-fluorescent proto-
porphyrin. It seemed therefore that a precursor in protoporphyrin
synthesis might be the substance we were looking for. We therefore
tried various concentrations of DAL incorporated in blood agar. A
series of plates of 7 per cent. blood agar containing various concentra-
tions of DAL were prepared. Three test organisms known to produce
red fluorescence in transplanted tumours were used (rabbit pasteurella,
Staph. pyogenes and E. cofi). Red-fluorescent colonies were produced
by all these organisms when an adequate concentration of DAL was
present in the medium. The optimum concentration appeared to be
approximately 0.05 per cent.

Experiment 15
It was found difficult to record photographicallythe red fluorescence
produced in the previous experiment, as the ultraviolet lamp used
could excite only a feeble red fluorescence when its beam covered
the entire surface of a culture plate. We therefore used a smaller
plate divided by a glass partition to illustrate this phenomenon (figs. 5
and 6). In the right half of this plate was placed blood agar and in
the left half 0.05 per cent. DAL blood agar. Both sides were seeded
with rabbit pasteurella. Fig. 6 shows that under ultraviolet radiation
the pasteurella colonies growing on DAL blood agar show red
fluorescence, which is absent from colonies on ordinary blood agar.

Experiment 16
In the previous experiment it was observed that the addition of
higher concentrations of DAL to blood agar changed the colour of
the medium from red to brownish red. This was no doubt due to
the formation of acid haematin in the medium through the action of
the HC1 present. It therefore had to be determined whether or not
this change in the medium had contributed to the production of red
J . PATH. m~ff.-vo~. 85 (1963) F2
86 F. N. GHADIALLY, W. J. P. NEISH AND H. C. DAWKINS

fluorescence. We also wanted to establish whether blood was necessary

in the medium for the production of red fluorescence.
Two organisms known to produce red fluorescence when injected
into tumours (Staph.pyogenesand rabbit pasteurella)and two organisms
incapable of doing this (Candida albicans and Str. pyogenes) were
selected as test organisms. Each of them was seeded on blood agar,
0.05 per cent. DAL blood agar, blood agar with equimolar amount
of HCI, serum agar, 0.05 per cent. DAL serum agar, plasma agar,
0-05 per cent. DAL plasma agar, chocolate agar, 0-05per cent. DAL
chocolate agar, nutrient agar, and 0.05 per cent. DAL nutrient agar.
Red-fluorescent colonies were produced by Staph. pyogenes and
pasteurella in all media containing DAL and some enriching material
(blood, serum, plasma, heated blood). These organisms failed to
produce red fluorescence in the remaining 7 media. No red fluorescent
colonies were produced by Candida albicans or Str. pyogenes in any
of the media.
Experiment 17
The results of experiments 14 and 15 indicate that DAL, a precursor
of protoporphyrin, can be converted by some organisms into proto-
porphyrin in vitro. It therefore had to be determined whether or not
these organisms could utilise the known precursors of DAL, i.e.,
glycine and succinic acid, to produce protoporphyrin in vitro. Amounts
of glycine and succinic acid equivalent to 0.05 per cent. DAL were
introduced into blood agar, and the same organisms as in experiment 14
were grown on this medium. No red-fluorescent colonies were
produced.
Experiment 18
A variety of organisms were tested on 0.05 per cent. DAL blood
agar for the production of red fluorescence in vitro. The following
organisms produced red fluorescence in vitro : Escherichia coli (++),
E. coli 0 111 K, 0 55 K and 0 26 K (+),Klebsiellapneumoniae (++),
Salmonella typhimurium and Salm. paratyphi B strain SU (+),Shigella
+ +
sonnei (+ +), Sh. flexneri (f +), Proteus vulgaris (+ +), Candidt
tropicalis (++), Neisseria meningitidis (j-),Staphylococcus pyogeiies,
4 strains (+++), Staph. albus (+), Staph. citreus (+++), Micro-
+
coccus tetragenus (+ +), Corynebacterium diphtheriae, types gravis
+
and mitis (+),Cl. hofmanni (+ +) and C. xerosis (++). The follow-
ing were negative in this test: Salm. paratyphi €3 strain CG, Salm.
typhi, Candida albicans and Cand. krusei, Neisseria cutarrhalis, Strepto-
coccus pyogenes groups A, B and C, Str. faecalis, Str. pneumoniae and
C . diphtheriae type intermedius.

Experiment 19
Four rabbits bearing ulcerated red-fluorescent squamous carcinomas
(3-6 cm. diameter) on the left ear and old and recent ulcers on the right
 RED FLUORESCENCENCE IN TUMOURS 87

flank were anaesthetised with intravenous Nembutal and the surface

of the tumours cleaned with soap and water till almost all the red
fluorescent material was removed. This procedure produced bleeding
and exudation from the tumour surface. Approximately 3 hr later
when gross exudation of fluid had ceased the animals were once more
inspected under ultraviolet radiation and a sketch made of each
tumour; the few small spots of red-fluorescent material that still
persisted in the grooved, lobulated surface of the tumour were noted.
Two of the 4 animals were now given protoporphyrin solution (one
5 ml., the other 10 ml.) via a vein in the right ear. The remaining
two rabbits not injected with protoporphyrin served as controls.
All lesions in control and experimental animals were inspected
under ultraviolet radiation at hourly intervals. Approximately 3 hr
after the administration of protoporphyrin all the lesions of both
experimental rabbits showed distinct but varying degrees of red
fluorescence. Fluorescence was stronger in tumours and recent ulcers
than in old ulcers, and more abundant in the animal that had received
the larger dose. The tumours of control animals showed no detectable
increase in fluorescence over that recorded in the sketches made after
cleaning the tumours; no ulcers shuwed red fluorescence.
At 6 hr all lesions in the experimental animals showed an increased
amount and intensity of red fluorescence. Hourly examinations were
abandoned at this point. At 24 hr the tumours of both experimental
animals were covered with an amount of red-fluorescent material far
in excess of any that we have witnessed on experimentally produced
squamous carcinoma in the rabbit in the absence of protoporphyrin
treatment. All ulcers also showed well marked red fluorescence. The
carcinomas of control animals, however, still showed no increase in
red fluorescence, and even 8 days later their red fluorescence, though
much increased, had still not quite reached the level present before
the tumours were cleaned.

Experiment 20
Efect of various doses of protoporphyrin in mice
Twenty mice bearing tumours and recent ulcers were given
intraperitoneal injections of protoporphyrin. Two received 1 mg.
of protoporphyrin each. Each successive pair received a dose
1 mg. greater than the previous pair so that each animal in the tenth
pair received 10 mg. of protoporphyrin. All surviving mice were
killed 20 hr later and their tumours and ulcers examined. Mice that
had received doses of 9 or 10 mg. were found dead and were excluded
from the experiment. No red fluorescence was detected in the lesions
of animals that had received less than 3 mg. of protoporphyrin. In
the animals that had received 3 mg. of protoporphyrin fluorescence
was detected in the ulcers but not in the tumours. In animals that
had received 4 mg. the ulcers showed more intense red fluorescence
88 F. N . GHADIALLY, W. J. P. NEISH A N D H . C . DAWKINS

than the tumours. With doses of 5-8 mg. both tumours and ulcers
showed brilliant red fluorescence.
The pattern of red fluorescence produced in this experiment was
different from that observed in previous experiments with experi-
mentally produced tumours (Ghadially, 1960) and with infected
tumours as described above. The fluorescence in the latter is restricted
to the necrotic areas, but in mice injected with protoporphyrin the
necrotic centres showed brilliant red fluorescence and the entire
tumour pink fluorescence.
Experiment 21
Persistence of fluorescence after protoporphyrin injection
Mice bearing Crocker carcinomas received injections of 5 mg.
of protoporphyrin. Pairs of them were killed at 1, 2, 3, 4, 5 , 8 and
11 days after injection and their tumours examined. Mice killed at
2 days showed more fluorescence than those killed at 1 day. A decline
in the degree of red fluorescence was observed in mice killed at 8 days
and practically none could be detected in those killed at 11 days.

DISCUSSION
These results demonstrate that red fluorescence can be produced
in tumours by injecting protoporphyrin into the host animal. On the
basis of this experimental model one might suggest that the phenomenon
of red fluorescence seen on human cutaneous carcinomas is also
perhaps due to the accumulation of circulating porphyrins on the
tumour surface. This possibility cannot be entirely ruled out, but
it is most unlikely that such a mechanism plays a prominent role in
the production of red fluorescence in human and experimental tumours.
If this were the only, or even the main, mechanism involved in the
production of red fluorescence, one would expect non-malignant
ulcerative lesions to show red fluorescence at least as frequently as
ulcerated squamous carcinoma. In fact, red fluorescence is rarely
seen in human ulcers and has not yet been observed in ulcers of the
experimental animal, unless the animal is given injections of proto-
porphyrin or also bears a squamous carcinoma (Ghadially, 1960).
Another point that militates against this hypothesis is that whilst
naturally occurring and infected tumours show red fluorescence only
in their necrotic parts, the entire tumour of the protoporphyrin-injected
animal shows reddish fluorescence. Two further criticisms can be
levelled against the hypothesis: first that a fairly large dose of proto-
porphyrin is required to produce red fluorescence in transplanted
tumours, and secondly that the fluorescence produced disappears
readily.
The bacterial hypothesis which we shall now discuss is more
acceptable since the fluorescence produced by bacterial agents is
persistent and shows the same distribution as that seen in naturally
 RED FLUORESCENCE IN TUMOURS 89

occurring tumours. Further, by this method red fluorescence can be

produced more readily and consistently in tumours than in normal
tissues.
The role of bacteria in the production of red fluorescence
The possibility that organisms may be involved in the production
of red fluorescence in tumours has been investigated in the past, but
with negative results. Ronchese et al. isolated organisms from many
cases of ulcerated human carcinomas on blood agar, but only in one
instance did they find a streptomyces that produced red-fluorescent
colonies. Sharvill failed to isolate such an organism in the many
cases he investigated and we too have failed to observe any red
fluorescence in the colonies of various bacteria isolated from human
and animal tumours. However the failure to obtain in-vitro red
fluorescence from bacteria isolated from the tumour surface cannot
be taken to indicate that they are not responsible for the production
of this phenomenon.
The present series of experiments demonstrates that if some of
the isolated organisms are allowed to grow in another tumour instead
of a culture plate they can reproduce the phenomenon of red fluores-
cence. Further we have observed that no specific organism is needed.
A variety of common organisms can produce this effect with varying
degrees of intensity. Since these organisms cannot produce red
fluorescent colonies when grown on ordinary laboratory media one
might suspect that the host or its tumour or both contribute some
essential material that is absent in the culture medium. That this is
indeed the case has been demonstrated by our in-vitro experiment in
which DAL was incorporated into blood agar. Further, we have
found a positive correlation between our in-vitro and in-vivo experi-
ments. Thus Staph. pyogenes, rabbit pasteurella and Escherichia coli
have all produced red fluorescence when injected into various trans-
planted animal tumours and also when grown on DAL blood agar,
whilst Str. pyogenes and Candida albicans have failed to produce red
fluorescence either in vitro or in vivo. Of the many organisms examined
to date we have not found one that produces red fluorescence with
one method and not with the other. It would therefore appear that
our in-vitro experiments reflect what is happening in man and experi-
mental animals.
We can now construct a hypothesis to explain most of the pheno-
mena associated with the red fluorescence of human and animal
tumours. It seems that the red fluorescence of tumours is due to the
production of protoporphyrin by the growth of a variety of organisms
on the ulcerated tumour surface, but for this reaction to occur the host
must provide a protoporphyrin precursor such as DAL and some un-
determined constituent present in the blood. It has been observed
that ulceration is essential for'the production of red fluorescence on
the squamous carcinoma of man (Ronchese et al., 1954). This is easily
90 F. N. GHADIALLY, W. J. P. NEISH AND H. C. DAWKI"

explained because it is under such circumstances that the tumour

becomes infected. Many transplanted animal tumours show red
fluorescence both when infected by natural ulceration and after experi-
mental injection of suitable organisms.

RedJluorescence of transplanted tumours

Policard (1924) reported that the necrotic centres of 4 experi-
mentally induced rat sarcomas showed red fluorescence when examined
under ultraviolet radiation. This finding was not confirmed by
Ghadially (1960), who failed to find red fluorescence in a variety of
transplantable rat tumours. This discrepancy can now be explained
if we assume that Policard's tumours were accidentally infected with
an organism capable of producing red fluorescence, whilst the tumours
used by Ghadially (1960) and by us in the present experiments were
protected from infection by the addition of penicillin and streptomycin.
To test this point we obtained a transplantable rat sarcoma and a
transplantable mouse Sarcoma from a department where tumours
are transplanted without the use of antibiotics. These tumours showed
red-fluorescent necrotic centres. The red-fluorescent mouse tumour
was minced with penicillin and streptomycin and transplanted by us
using our usual technique. None of the daughter tumours showed
red fluorescence. We have also tested the penicillin and streptomycin
sensitivity of the organisms capable of producing red fluorescence
isolated from rabbit and human carcinoma and have found that they
are sensitive to one or both drugs.
Thus it would appear that a non-ulcerated transplantable tumour
that shows a red fluorescent centre is an infected tumour. This simple
test we feel might be of some practical value to those using transplanted
tumours. The absence of red fluorescence cannot, however, be taken
to indicate that the tumour is free from all infection, for it might carry
an organism incapable of producing red fluorescence.

Red fluorescence in non-malignant lesions and normal tissues

A point that has led to much controversy in the past is whether
red fluorescence is seen on ulcerated human squamous carcinoma
specifically or can also occur on ordinary chronic ulcers, for Shanrill
(1955) claimed that red fluorescence can occasionally be detected on
non-malignant chronic ulcers of man.
Belisario (1959) after reviewing the available literature on this
point concluded that this claim is only partly, if at all, admissible
and that the presence of the characteristic fluorescence, on the informa-
tion and findings available (including his own), is still reasonably strong
presumptive evidence of the presence of squamous carcinoma.
The experimental results obtained by us do not help to resolve
this dilemma, for here we are faced with a species difference. In the
mouse and the rabbit red fluorescence can be produced in normal
 RED FLUORESCENCE IN TUMOURS 91

muscle, but this is not so in the rat, hamster and monkey. It is

interesting to note, however, that tumours in all these species, as well
as those of man, show red fluorescence when infected with certain
organisms. These results recall Greenstein’s observations that tumours
more nearly resemble each other chemically than they do normal
tissues or than normal tissues resemble each other, and that the
metabolic pattern of tumours is similar in all species studied. This
then might explain why red fluorescence is so readily and constantly
produced in a variety of tumours in various animal species, but is
only occasionally seen in non-malignant lesions or normal tissues of
some species.

Fluorescence of squamous and basal-cell carcinomas

A point of considerable biological interest is that red fluorescence
is seen on the surface of ulcerated squamous carcinomas but not
basal-cell carcinomas (Ronchese et al., 1954). We have now estab-
lished that red fluorescence is produced on the surface of human
carcinomas by the growth of common organisms like Staph. pyogenes
and E. coli. It is difficult to believe that these organisms can be so
specific in their needs that they can grow on squamous carcinoma
but not on basal-cell carcinoma. A more likely explanation is that
these common organisms occur on a variety of lesions besides squamous
carcinoma, but either some essential factor contributed by the
squamous carcinoma or its host is lacking, or there is some factor
present in the basal-cell carcinoma that inhibits this reaction. It seems
to us that though the presence of a suitable organism is an important
factor in the production of red fluorescence it is certainly not the
only factor involved.

SUMMARY
Protoporphyrin was injected into animals bearing experimentally
induced tumours, transplanted tumours and non-neoplastic ulcers.
It was found that by this method red fluorescence in ultraviolet light
could be produced in ordinary ulcers at least as readily as in tumours.
It seems unlikely that such a mechanism is involved in the production
of red fluorescence in human cutaneous squamous carcinoma since
red fluorescence is rarely seen in chronic ulcers of man, but is almost
always detectable in ulcerated squamous carcinoma.
The necrotic centres of transplantable tumours of mice and hamsters
show red fluorescence in ultraviolet light when these tumours are
infected with certain organisms such as pasteurella, Staphylococcus
pyogenes and Escherichia coli isolated from the surface of ulcerated
squamous carcinoma of rabbit or man. It would seem therefore
that the red fluorescence of ulcerated human squamous carcinoma
is due to microbial activity on the surface of the tumour. Red fluores-
cence was also produced in the muscles of some of the mice and rabbits
92 F. N. GHADIAUY, W. J. P. NEISH AND H. C. D A W K l "

injected with cultures of these organisms but not in those of the rat,
hamster and monkey.
Organisms capable of producing red fluorescence in human,
experimentally induced and transplanted tumours will produce red-
fluorescent colonies when grown on blood agar to which S-amino-
laevulinic acid (DAL) is added.
These experiments suggest that the red fluorescence of tumours
may be due in part to the action of bacteria on a protoporphyrin
precursor such as DAL and on the blood of the host.
We are indebted to MIS J. White for technical assistance. This work was
supported by a grant to one of us (F. N. G.) from the University of Sheffield
Medical Research Fund.

REFERENCES
J. C. .
BELISARIO, . . . . . 1959. Cancer of the skin, London, p. 76.
GHADIALLY,F. N. . . . . . 1958. This Journal, 75,441.
9, . . . . . 1960. This Journal, 80,345.
F. N.,
GHADIALLY, AND NEISH,1961. Nature (Lond.), 188, 1124.
W.J. P.
H.,A N D P A m , A.
GOUGEROT, . .
1939. Bull. SOC.franc. Derm. Syph., 46,
288.
O., AND GHADIALLY,
ILLMAN, F. N. 1960. Brit. J. Cancer, 14, 483.
POLICARD,
A. .
. . . .
. .
1924. C.R. SOC.Bwl. (Paris), 91, 1423.
RONCHESE,F., WALKER,
B. S., AND 1954. Arch. Derm. Syph. (Chicago), 69,31.
YOUNG,R. M.
D. . . .
SHARVILL, . .
. . 1955. Trans. St John's Hosp. Derm. SOC.
(Lond.), no. 34, 32.