Apoptin gene therapy in head and neck cancer Schoop, R.A.L.
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(2) 4 A mouse model for oral squamous cell carcinoma. Remilio A. L. Schoop . . .
(3) Robert J. Baatenburg de Jong. ! " .
(4) Abstract Despite recent advances, the prognosis of oral squamous cell carcinoma is still poor. Therapeutic options such as radiotherapy, chemotherapy, surgery and the novel treatment option gene therapy are being investigated in animal models. Diverse models have been studied to induce oral squamous cell # $ % & % '( )
(5) *+- # ( proven to be successful although until now it is unknown at what time point the established tumor is a representative squamous cell carcinoma and has / # 0# 1 (233(
(6) *+4 times a week during 16 weeks and measured the volume of tumor tissue each 27 ( '3# 27 8 3 ( # 3 3 ( '3# /( mice bearing oral tumors were diagnosed with squamous cell carcinoma. Immunohistochemistry with markers cyclin D1 and E-cadherin revealed that the generated mouse oral tumors showed strong similarities with the described immunopathology in human oral tumors. $
(7) *+ # ( / 3 3 '3# 2 3# oral tumors. 1 / 3 3
(8) *+ model should be conducted around 40 weeks after the start of the treatment.. 58.
(9) Introduction Oral cancer accounts for almost 275,000 new cases worldwide each year1. The main risk factors for developing oral cancer are tobacco smoking and consumption of alcohol2. Continuation of smoking during treatment can lead to increased morbidity and mortality4. Primary treatment is associated 2# (( ?2 3 @F(( recurrence4. Present-day treatment, surgery, radiotherapy or chemotherapy or a combination of these three, has had notwithstanding the improvements, 0 #3 /# //5. 1 2 #? )(-3(/ 3(6-10. New therapies can be investigated both in-vitro and in-vivo. The drawback of in-vitro laboratory study is the dissimilarity between the cell culture and the physiological processes giving misleading results. Several animal models for oral squamous cell carcinoma are used including hamster, rat and mouse models, with each model having its own advantages and disadvantages. G'3#2 ( # # damaging the jaw in mice11. Nowadays ' # ( 2( ( H# ( #K(( 2## (0# Advantages are the speed and certainty of tumor development. The premier limitation is the lack of functional T lymphocytes in nude mice resulting in a non physiological tumor response. The. widely. used. hamster. cheek. pouch. model. in. which. ? %(#M)- ( ( # ((/ because the tumors are unalike the human counterparts12. After the introduction of %
(10) & % % '( )
(11) *+- ( # rats 4?'/ ( (# ( analyzing the effects of anti-tumor agents14. $
(12) *+ & # carcinoma model is a lengthy multi-step process in which eventual invasive squamous cell carcinoma is attained after several months. Once established the tumors are suitable for studying the therapeutic. 59.
(13) effect of various therapies including gene therapy. Intratumoral injection of recombinant adeno-viral vectors is mainly used in preclinical models15. Therefore at the time of injection a minimum tumor volume must be attained and the realization of squamous cell carcinoma must be assured. Up till now,
(14) *+# # (?#3 7 2 The present study was set up to investigate the time needed for 0 # / # (/ 3(% # K 3 Q#3 ?2/(0( the generated mouse oral tumors resembles human oral tumor formation.. Material and Methods Animals and treatment with carcinogen. Thirty-two 7 and 8 weeks old male CBA mice weighing between 4 ( 2 3 ( # W? $
(15) ( $ '3# 2 33 /( (/ X( Y/ Q# Z8 ## Q#273(( ( of alternating 12-h periods of light and darkness and were fed sterilized laboratory chow and water ad libitum. The carcinogen 4-Nitroquinoline-1 '()1 -2( /(3 3 0 @#[# 1 & 2 ( Q2/( by inhalation of halothane vapour and the tongue was stroked once with a no 4# 16?2((33(
(16) *+ $# 2 ( # (7 0
(17) *+ 33 $ #2(2
(18) *+( \272& 427 treatments. All mice were carefully inspected daily and weighed weekly.. 60.
(19) Figure 1 8# % & % '()
(20) *+-. Pathological Examination and Immunohistochemistry. Q0
(21) *+33 # (/( # 2'#( ( /27 $/ # ' 3 # 2 (27# (# )? ( 2(- Q 27 2 '3#\#20(( 2# /((0'( #( F ( # ] & 2 3 \ #0("?4?4\( 27 Q30%#((? ^m width were made ( 0/ & 2 ( 2 # '% ) G- $ 3 2 '#( 3 ( 0( # 3? #( (3? # ( (3? sever dysplasia or invasive squamous cell carcinoma. Dysplastic epithelia and invasive squamous cell carcinoma were subsequently investigated by immunohistochemistry with cyclin D1 and E-cadherin. The following antibodies were used from Santa Cruz Biotechnology, H )]8 M?8Q-: 3 ( ` ) %@? ( - and a rabbit polyclonal antibody against G%() % "?( @- Q(2(30M((((? (233( according to the manufacture’s protocol.. 61.
(22) Results
(23) $0# 32 27 '3# Q 27 # (( 2 3 )1 Q- #3332 3 (#((32(()1 x- 1 27 ? (/32 ((# ()1 8-(/(3 )1 `- ((( $0/( & # # 2(0(427)1 G- Q#3 4\27 \ # 2 0( 2( & # # 3 '# Q 27?( '3#?\ # 2& # #)( 2- Figure 2 A. B. normal; 24 weeks. mild dysplasia; 24 weeks. C. D. moderate dysplasia; 28 weeks. severe dysplasia; 28 weeks. E. invasive squamous cell #427. 62. 0( # ( 2
(24) *+ ( \ 27 )Q-
(25) # 2 histopathological changes 24 weeks after starting the '3# )x- ( (3 27 )8- ( (3 " 27 )`- ]/ (3"27 )G-H//& # # 4 27 + #0 ' .
(26)
(27) Once all 6 mice bearing tumors at the 40 weeks time point were diagnosed with squamous cell carcinoma, immunohistochemistry was 3 #( Q #3 8` (G%('3 2#( #( (3 ( 27 )1 4Q- ( & # # (( 27 )1 4x- H## evaluation was done in the tumors of our interest. Figure 3 A. B. mild dysplasia; 24 weeks. squamous cell carcinoma; 40 weeks. C. D. cyclin D1; 24 weeks. cyclin D1; 40 weeks. G. . . E-cadherin; 24 weeks. . . 1. E-cadherin; 40 weeks. 3 ( ## # staining with biomarkers cyclin D1 and E-cadherin. The left panel shows mild dysplasia after 24 weeks, the right panel shows squamous cell carcinoma after 40 weeks # '% ) G- #((3 )Q-( & # #)x- 2 8` 3#'3( #( (3 )8- ( throughout the tumor and in tumor nests in squamous cell carcinoma )`- G%('3(3 3 #? #( (3 )G- ( 3 lost in the invasive front of & # #)1- +#0 ' . 63.
(28) 8` '3 2/( antigen. Only nuclear staining was considered positive, while cytoplasmatic 2 (( / ( (3 '3 ( 3( # '3` )1 48- $ stainability increased with the progression from dysplasia to carcinoma and the positive ratio was the highest in the & # # )1 4`-. After 40 weeks, squamous cell carcinoma showed a distribution throughout the tumor )1 4`-2( 23 ] 27 2(G%(2 '3( 3? )1 4G- H & # #2 ( G%('3 // (3 / )1 41- $ # # / # ' 3 # 2 ( 2 27 2 '3# 3 ( By measuring the size of the tumor in three dimensions the volume was calibrated in mm4 by using the following formula: V = l x w x h. The volumes ( #3 21 Q27? average volume was 6 mm4 with a range of 2 to 12 mm4. Gradually with time / #(((4##4 at the 40 weeks time point.. volume in mm4. Figure 4 45 40 35 30 25 20 15 10 5 0 20. 24. 28. 32 weeks. 64. 36. 40.
(29) Discussion Oral squamous cell carcinoma is one of the most common cancers in the world. Novel treatment is best studied in animal models that mimic the same clinical features as human squamous cell carcinomas. In this study we induced oral squamous cell carcinomas by applying
(30) *+ ( \ 27 # }# 33( 4 27 33
(31) *+ $ 3 # 3 for squamous cell carcinoma was seen before invasive squamous cell #33( Q4\272 ( # 2& # cell carcinomas, 4 weeks further all 6 oral tumors were squamous cell carcinomas. These results are in accordance with earlier observed results in 2 & # # 2 (( 2 ( 44 27 33
(32) *+( \2716,17. Immunohistochemistry was performed on the tissues to assess the similarities with human squamous cell carcinoma. Biomarkers have been studied to predict behavior of tumor cells and are used to characterize tumors18. Cyclin D1 plays a pivotal role in transition from G1 to S phase and ` '3 3 ( # ( //19. As is seen in human and other animal models20,21 ` '3 2(('3 & # # mice. E-cadherin, a calcium dependent membrane protein that is essential for the formation of adherens junction between cells. There is a connection 2'3 G%(( #33 to invasive carcinoma22 Z /( '3 G%( // #(/( ('3 stages, a pattern that has been reported earlier in human oral squamous cell carcinoma4. Average tumor volume advanced from 6 mm427 4 mm4 27#3 1 % # K 2( / (M ' # ##? . 65.
(33) preferable higher. Eventual tumor size reduction after treatment is then Q #
(34) *+ # # (2 /3 3 to develop in to a large tumor volume such as the model in which tumor cells K( 7? # / #2 '( 100 mm4 }/ '3#2% # K 3 2(K(/ ## 34F / # tumor24. Taking this in to account and knowing that the volume of an oral squamous cell carcinoma in mice is small we estimate that a tumor volume of at least 15 mm4( Q427 # / # 3( 3 ( 27 / # /( 4 ##4 Z # that a desirable tumor volume will be reached after these 40 weeks and the histopathology will reveal a squamous cell carcinoma, without hampering the animal’s welfare. $2 # & ('3#3 ( / 3%/ ( (# $
(35) *+# ( / (/ ( ((/ / '3# Compared with the cell lines and the nude mouse model, the close similarity
(36) *+# ( 3 3 0 $ drawback of the nude mouse model is the lack of an immunocompetent component. The weakness of the hamster cheek pouch model is the earlier mentioned dissimilarity of the tumors with the human equivalent. Q33 `xQ ## 3 ( #7 it hard to study early squamous lesions25
(37) ## necrosis was seen in our specimens. Other drawbacks of this model are that the cheek pouch has no anatomic counterpart in human, the epithelium of 73 0 3 # of humans and mice and the tumors seem to progress from papillomas26,27, which is uncommon in humans and was not seen in our samples. The clear ((/
(38) *+ # ( ( ( # # #('3# and most of the time readily available. 66.
(39) In our conclusion taking both advantages and disadvantages in to
(40) *+ # # (0(# for therapeutic research applications.. 67.
(41) References }7`?x1?1?}} ? 8Q Cancer J Clin 2005; 55: 74-108. x }? Q ( X+ \ %@\ 4 (?]XZ?`Q? ZH?$ #7Q$?$ }$? `? GZ 8 (# 73/ chemoradiotherapy for limited-stage small-cell lung cancer is associated with (( // 8+ 4 @ % 8?1/]?$##]?x ?# (G G(3 2 agents in induction chemotherapy for locally advanced head and neck cancers Q + 4 @% \ @ 8 3]?}K7$1?1 QQ? ?8#3x ?]'#]x? Q?# G?8#7Q? #??G1?8 ]?] M 8?X
(42) ?1 } 3/ ( 3(# 3 for high-risk squamous-cell carcinoma of the head and neck. N Engl J Med 4@ 4 % \ H? ZM $G? Q(K QQ $ 3 3 32 therapy. CA Cancer J Clin 2005; 55: 178-94. ] 3Q? ?x ( ?
(43) x%'X 3@4% 3 3%( ( 3 3 ( ( 7 & # # H8 4"% " ] 3Q?x ( ?
(44) Q3 3( 3 3 # # ( 8x $" 4\"% 4 X %?]2H?]3M?W2M G1 3 ((7& # # G'3+3H/ ` " @ %4 10. Ganesh S, Gonzalez-Edick M, Gibbons D, Ge Y, Vanroey M, Robinson M, Jooss K. Combination therapy with radiation or cisplatin enhances the potency of Q(@[4@# ( / 3# ( ((7 cancer. Cancer Gene Ther 2008. G (7 1 G'3# ## K2 # #
(45) ( $K( 7( \ 4% K `? ( % & % % '( ( ( '3# carcinogenesis. Oral Oncol 2006; 42: 655-67. 4 Z ? X7 # Y + ( ( 2% %
(46) % '( +( / 4 4% " ] M 7? ?] M ? #
(47) ?$ ( ?Z7?$7$ Q## ( 3(( 3# #% % 3 % % & % '( 3 3# 3/ ( 8 \ \ %4. 68.
(48) 15. St George JA. Gene therapy progress and prospects: adenoviral vectors. Gene $4 4@% \ 27 xX? ( xZ? Q7# `? X ? M ]Q?. ( 1
(49) *+ # # ( / & # # (
(50) 7 \ %4 $ ? (x?x#`?$7 ]? (X +/( 3 carcinogenesis modeled in carcinogen-treated mice. Clin Cancer Res 2004; 4 % 4 18. Takes RP. Staging of the neck in patients with head and neck squamous cell cancer: imaging techniques and biomarkers. Oral Oncol 2004; 40: 656-67. x /?* `H?
(51) 7/]?8 HG?](x1?? ? 8?] (X 8` (3 \H
(52) Q'3 3( reduced survival in carcinoma of the anterior tongue. Clin Cancer Res 1999; 5: 2810-9.
(53) 2]?Y ]?] Q 3'3 (/ 3# #( ( % & % '( ++ 4 @ %"@ ( ? $7 $? ? # 1? ? $ ( ? Q? ] ]?]$? ` % & % '(% ( ( 8X@ %4 } Q? Z }? ` Y? ]# ? 8 Q G%( #( # #
(54) "4 %4 4 Z# ?](`]?7 27Q?X(?x 2Q G'3 of cadherins and catenins in oral epithelial dysplasia and squamous cell # +} ( " 4"% /(G?}Q?]3K1? 33}?/(??
(55) ? 8 323 3( ' ( #3 # 8$ @4%\ @
(56) ? ( X?
(57) 77 }? ZK ? # Q G3 (3 ( & # # Z 3 #
(58) *+# ( (
(59) 7 \ " % \
(60) ? ( X?
(61) 77}?ZK?#Q 8 #3 3(3%%
(62) *+3# (( # # H +' ] @ @4%" 27. MacDonald DG. Comparison of epithelial dysplasia in hamster cheek pouch carcinogenesis and human oral mucosa. J Oral Pathol 1981; 10: 186-91.. 69.
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