17385182

LUNG TUMOR LOCATION AND LYMPHOCYTE INFILTRATIONIN MICE ARE GENETICALLY DETERMINED

Hugo Horlings & Division of Pathology, Netherlands Cancer Institute, Amsterdam,The Netherlands

Peter Demant & Division of Molecular and Cellular Biology, Roswell Park CancerInstitute, Buffalo, New York, USA

& Lung cancer is a heterogeneous disease with individual differences in histological type, rate ofprogression, and response to therapy. Definition of the molecular and genetic basis of specific tumorcharacteristics would provide a better assessment of prognosis and a basis for a more individualizedtherapy. Here the authors compare the quantitative and qualitative phenotypes of lung tumors inmice of O20=A and OcB-9=Dem strains subjected to 2 regimens of N-ethyl-N-nitrosourea (ENU)treatment: (1) prenatal tumor induction by a single intraperitoneal (IP) injection of 40mg=kg bodyweight into pregnant females and (2) after the same prenatal induction, the progeny received onweeks 9 and 11 additional IP ENU injections. The numbers, size, and histological characteristicsof tumors were determined microscopically in semiserial lung sections. Unexpectedly, the authorsobserved very highly significant strain differences in a novel polymorphic phenotypeperibronchialversus nonperibronchial location of lung tumors, as well as in frequency of lymphocyte infiltration.To assess the reproducibility of these genetic differences, the authors classified both tumor locationand lymphocyte infiltration also in an independent set of lung tumors that were induced in thesestrains in experiments performed more than 10 years ago in a different mouse facility and foundthe same strain differences. These results indicate that these qualitative phenotypes are very robust(Pc 5.52 106 and 2.27 108, respectively) and relatively independent of environmentalinfluences. They likely reflect different stages of lung differentiation at the time of tumor inductionand differences in molecules involved in intercellular signaling, respectively. The definition ofgenes controlling these traits will provide novel insights into the determination of tumor phenotype.

Keywords lung tumors, lymphocyte infiltration, mouse, peribronchial location

Received 26 August 2004; accepted 21 October 2004.This work has been supported by grants to P. Demant from the European Commission (PL98-0445:

Identification of Genes Responsible for Multigenic Control of Disease Susceptibility, and ERBF CT-98-0317: Genetic Dissection of Multigenic Predisposition to Disease in the Mouse Model) and from TheHealth Research Institute, Buffalo, New York.

Address correspondence to Peter Demant, Roswell Park Cancer Institute, Elm and Carlton Streets,Buffalo, NY 14263, USA. E-mail: [email protected]

Experimental Lung Research, 31:513525, 2005Copyright # Taylor & Francis Inc.ISSN: 0190-2148 print/1521-0499 onlineDOI: 10.1080/01902140590918740

Lung cancer is a major public health problem worldwide and accounts formost cancer deaths among both men and women [13]. Although 90% oflung cancer in women and men in the United States [4] can be attributedto cigarette smoking, there is strong evidence for genetic susceptibility andgene-environment interactions in the development of lung cancer [57].The major predictor of clinical behavior of human cancer has been its his-tological appearance. Presently, gene expression profiles can be comparedwith already known tumor types and classes, or indicate the existence ofnew ones [813]. New morphological and biological descriptors arerequired for qualitative assessment of tumor types [14] that will matchand validate the emerging expression clusters.

Segregating crosses between susceptible and resistant mouse inbredstrains or recombinant congenic strains had been used to detect and mapa large number of susceptibility genes to lung tumors [1519]. Most of thesestudies have focused on measurement of lung tumor number, size, or theircombination (tumor load). However, lung tumors within a mapping crossdiffer also histologically and different mouse inbred strains develop prefer-entially different tumor types, indicating that hosts genes influence qualitat-ive aspects of mouse lung tumors as well [14, 20, 21]. The genes specific fordifferent type of mouse lung tumors (alveolar, papillary, and mixed) werepostulated but could not be mapped [22], perhaps because this phenotypeclassification, although biologically relevant, does not sufficiently accountfor the underlying biological and genetic complexity of these phenotypesand may be confounded by presence of various not readily distinguishableintermediate phenotypes. On the other hand, genes controllingmore simpleunit phenotypes, such as 3-dimensional shape of tumors and presence ofnuclear cytoplasmic inclusions, have been readily mapped [14, 23].

The O20-congenic-B10.O20=Dem (OcB=Dem) recombinant congenic(further RC) strains carry each a different random set of 12.5% genes ofthe strain B10.O20=Dem (B10.O20) and the remaining genes from thestrain O20=A (O20) [24]. They are suitable for study of the genetics of quan-titative and qualitative aspects of lung tumorigenesis, because their two par-ental strains, O20 and B10.O20, differ in susceptibility to lung cancer aftertreatment by carcinogen N-ethyl-N-nitrosourea (ENU): O20 mice developmore and larger tumors than B10.O20 mice, but also develop consistentlydifferent histological types of lung tumor [25]. The O20 lung tumors areoften mixed alveolar (in the periphery) and papillary (mostly in the center)with abundant nuclear pleiomorphism, whereas B10.O20 mice lung tumorsare papillary, with no or very little pleiomorphism [21]. OcB RC strains con-tain different mixtures of O20 and B10.O20 genes and display interstraindifferences in both the number of tumors and tumor size, but also exhibitan exceptional variety of tumor types [25]. Morphological evaluation oflung tumors in OcB strains revealed also extensive differences in tumor

514 H. Horlings and P. Demant

shape and in presence and extent of nuclear pleiomorphy, lymphocyteinfiltration, and tumor heterogeneity [14, 23, 26].

In order to obtain further insight into the genetic and molecular basisof lung tumor progression and to define the correlations between the hostgenotype and various aspects of tumor phenotype, we assessed the qualitat-ive phenotype of mouse lung tumors induced by different doses of carcino-gen (ENU) using 12 unit phenotypic characteristics that are unequivocallyidentifiable under the light microscope (see Materials and Methods) andevaluated them in the lung tumors from the strain O20 and the recom-binant congenic strain OcB-9.

Compared to previous work fromour group [23, 25], we present evidencefor a surprising novel unit phenotypelocalization of tumors in relation tothe bronchial treeand show that it differs considerably between the strainsO20 and OcB-9. We show also a significant difference in presence of tumor-infiltrating lymphocytes between these strains. The location of the tumors inrelation to the bronchial tree has not been published or analyzed before, andto the best of our knowledge there are no previous reports of strain differ-ences in this phenotype. After this unexpected finding, we classified boththe tumor location and the lymphocyte infiltration also in archival histologi-cal slides of lung tumors induced and processed in the same way as in thepresent study in the O20 and OcB-9 strains in an independent set of experi-ments that were performed more than 10 years ago in a different mousefacility and we found the same strain differences. The identical quantitativetumor phenotypes observed in the 2 strains after many years, in spite of com-pletely different animal facility, geographical location, and experimentalteam, demonstrate that these phenotypes are robust and reproducible.

MATERIALS AND METHODS

Mice and Carcinogen Treatment

The mouse strains for this study were shipped from The NetherlandsCancer Institute in Amsterdam to State Pharmacology Research Institutein Rosice, Czech Republic, where they were bred for the experiments. Micewere maintained under a strict light-dark regimen and received acidifieddrinking water and a standard laboratory diet ad libitum. At day 18 of ges-tation, pregnant mice were injected intraperitoneally with 40mg=kg bodyweight of ENU (Serva, Heidelberg) dissolved in phosphate-buffered citricacid (pH 6) [14, 15, 17, 19]. Eleven mice of the O20 strain and 15 of theOcB-9 strain received only the transplacental ENU treatment (further1ENU group). Fourteen O20 mice and 17 OcB-9 mice received inaddition to the transplacental treatment 2 intraperitoneal injections of ENUon weeks 9 and 11 after birth (further 3ENU group). All offspring of

Location and Lymphocyte Infiltration of Lung Tumors in Mice 515

injected mothers were euthanized at 16 weeks of age and their whole lungswere removed, fixed in AEF solution (i.e. 40% v=v ethanol, 5% v=v aceticacid, 4% v=v formaldehyde, and 0.41% NaCl) and embedded in histowax.The lungs were sectioned semiserially (5-mm sections at 100-mm intervals)and stained with haematoxylin-eosin. The Animal Experimentation EthicsCommittee of the Netherlands Cancer Institute has approved the set-upof the experiments.

Lung Tumor Size and Load

Lungs were sectioned semiserially at 5-mm intervals and every 20th sec-tion has been stained by haematoxylin-eosin and examined microscopically.In most cases we obtained and examined 30 to 35 such sections per mouse.The lungs were scored for number of tumors, tumor size, and tumor loadas described previously [15]. Tumor size is expressed as the sum of all mea-sured surfaces of the tumor (calculated with the aid of a grating in the ocu-lar) in the successive semiserially sections. The tumor load is expressed asthe sum of sizes of all tumors in a mouse. Only tumors greater than 300-mmin at least one section were included.

Morphology

Histological evaluation had been performed in parallel by 2 observers. Abinary (presence=absence or yes=no) system of scoring was used for all 12qualitative variables. These are (a) peripleural location (contact with pleura)versus nonperipleural, (b) peribronchial location versus nonperibronchial,(c) papillary structures, (d) alveolar structures or (e)mixed (alveolar and pap-illary) tumors, (f) lymphocyte infiltration, (g) blood vessels peripheral to thetumor, (h) centrally located blood vessels, (l) nuclear cytoplasmic invagina-tions, (m) nuclear pleiomorphy, (n) mitoses, and (o) intratumor heterogen-eity. Tumor location has been classified in 2 different ways: first, peripleurallocationwhether a tumor is located in the periphery of a lung (direct con-tact of part of tumor cells with pleura) or not, and second, peribronchialpositionthe tumor is growing next to or around bronchiolar structures(direct contact of part of the tumor cells with the bronchial wall or epi-thelium) or not. Infiltration of lymphocytes in the tumor was defined bythe presence of 15 or more lymphocytes in at least one semiserial sectionof a tumor. Many tumors did not contain any obvious infiltrating lymphocytesat all, whereas others contained several tens or hundreds lymphocytes in oneor more sections. Assessment of tumor heterogeneity was based on identifi-cation of distinct areas with different cellular morphology and nuclear atypiawithin a tumor (presence of both alveolar and papillary structures in the sametumor region did not fall under this criterion).


Statistical Analyses

The quantitative features were evaluated by Mann-Whitney test, and chi-square (v2) tests were performed for the qualitative features. The obtainedP values were corrected for multiple testing by the Bonferroni correctionby multiplying them by 12 (the number of parameters tested) to obtainthe corrected Pc values.

RESULTS

Quantitative Characteristics

The quantitative aspects of lung tumors of strains O20 and OcB-9 arepresented in Table 1. O20 mice are generally more susceptible mice thanOcB-9 mice. Significant differences were between the tumor load of O20and OcB-9 mice (pooled sexes and experimental groups, Pc :0053).The rest of the strain differences are not statistically significant, but almostall measurements point to a higher susceptibility of the strain O20.

Qualitative Characteristics

Frequency of individual histopathological qualitative characteristics oftumors in O20 and OcB-9 mice is summarized in Table 2. There was no sig-nificant difference between the O20 and OcB-9 mice in the following char-acteristics: peripleural (pleurally located) versus nonperipleural location;frequency of alveolar, papillary, or mixed tumor type; and peripheral andcentrally located blood vessels, nuclear cytoplasmic invaginations, nuclearpleiomorphy, detected mitoses, and tumor heterogeneity.

TABLE 1 Quantitative Phenotypes of Lung Tumor of Strains O20 and OcB-9

1ENU 3ENU

O20 OcB-9 O20 OcB-9

Number of mice 11 15 14 17Females=males 4=7 8=7 5=9 6=11Number of tumors 22 18 22 13Number of tumors in females 9 4 11 6Number of tumors in males 13 14 11 7Mean number of tumors per mouse 2.0 1.2 1.6 0.8Mean number of tumors per female mouse 2.2 0.5 2.2 1.0Mean number of tumors per male mouse 1.8 2.0 1.2 0.6Mean tumor size 21.1 4.3 34.7 3.5Mean tumor load 107mm3 per mouse 42.3a 5.2a 54.5a 2.7a

aTumor load in O20 mice is higher than in OcB-9 mice (P .0053).


Two qualitative phenotypes exhibited a striking strain difference. Lym-phocyte infiltration is significantly more frequent in O20 than OcB-9 mice(v2 17:18292; Pc :00041; Table 2). A similar observation has been alsomade by Tripodis and Demant [23] analyzing the tumors induced in theperiod 19901994 at The Netherlands Cancer Institute in Amsterdam.Pooling the data from these 2 independent experiments reveals that 58out of 100 O20 tumors, but only 7 out of 70 OcB-9 tumors, contained infil-trating lymphocytes. This difference is highly significant (v2 40:17;Pc 2:27 108; Table 3).

Surprisingly, we observed a large difference in location of the lungtumors (Figures 1 and 2) in the two strains. The tumors from the O20 miceare more frequently (15 out of 44) than in OcB-9 mice (1 out of 31) locatednext to or around bronchiolar structures (v2 10:32379; Pc 0:0156;Table 4). To control the validity of this observation, we analyzed the tumorlocation in the independent set lung tumor of O20 and OcB-9 mice that

TABLE 2 Qualitative Characteristics of Lung Tumors in Strains O20 and OcB-9

O20 OcB-9

No Yes No Yes v2 P Pc

Peripleural location 32 12 18 13 1.7595 .1846 n.s.Peribronchial location 29 15 30 1 10.3238 .0013 .0156Lymphocytic infiltration 21 23 29 2 17.1829

were induced at The Netherlands Cancer Institute in the period 19901994[25]. In this material we also found that O20 mice develop peribronchialtumors frequently (15 out of 27), but OcB-9 mice not (none out of 20)(v2 16:3190; Pc 6:422 104; Table 4). The 2 sets ofO20 andOcB-9micecombined (122 tumors) showed highly significant difference v2 25:4237; P 4:6046 107; Pc 5:52 106; Table 4). We did not detectany significant differences between the peribronchial and nonperibronchial

FIGURE 1 A peribronchial tumor from an O20 mouse treated prenatally with ENU (A: 100magnification; B: 400magnification).

FIGURE 2 A peribronchial tumor from an O20 mouseclose contact of tumor tissue with bronchialwall (400magnification).


TABLE4

CorrelationofTumorLocation(Peribronch

ialversusNonperibronch

ial)withStrain

O20

andStrain

OcB

9Tumors

Rep

orted

inReferen

ce15

(n47),ThisStudy(n75),an

dtheTwoSetsCombined

(n122)

withv2,P,

Pc

Site

andyear

of

experim

ents

O20

OcB

-9

Nonperibronch

ial

Peribronch

ial

Nonperibronch

ial

Peribronch

ial

Total

v2P

Pc

Czech

Rep

ublic,

2000

2915

301

7510.2328

.0013

.0156

TheNetherlands,

19901994

1215

200

4716.3190

5.35210

5

6.42210

4

Total

4130

501

122

25.4237

4.60510

7

5.52610

6

520

tumors in histological type, size, or other characteristics, although they mayappear when more tumors are tested.

DISCUSSION

Transplacental exposure to ENU at day 18 of gestation and weeks 9 and11 after birth results in both quantitative and qualitative differencesbetween lung tumors in O20 and OcB-9 mice. These results are in agree-ment with the previous results indicating a higher susceptibility of O20than B10.O20 mice (for review see reference 25, 26). In the present resultsthis has been reflected in the differences in tumor load and tumor size(Table 1). Interestingly, additional postnatal treatment with ENU did notalways result in larger tumor number, load, or size in either strain.

Paradoxically, in OcB-9 mice the additional ENU injections do notappear to lead to increased tumor size and load (data not shown). Possiblythe apoptotic and toxic effects of ENU [27, 28] known to affect also rodentalveolar epithelial cells [29] temporarily prevail over the effects of thosenovel somatic mutations that could lead to a faster tumor growth and pro-gression. This growth-enhancing effect might require a longer time tobecome obvious. On the other hand, a certain, although not significant,increase of tumor load and tumor size is seen in O20 mice, possibly indicat-ing a genetic difference in inducibility of apoptosis or susceptibility to toxiceffects of ENU.

The observed strain differences in the 2 qualitative tumor phenotypesperibronchial location and lymphocyte infiltrationexhibit an impressiverobustness. They were observed and confirmed in 2 different experiments,carried out 10 years apart in 2 animal facilities, in Amsterdam, theNetherlands, and in Rosice, Czech Republic. This indicates that thesephenotypes reflect intrinsic properties of the 2 strains rather than randomenvironmental effects on tumorigenesis.

The differences in location of the lung tumors between the strains O20and OcB-9 are remarkable. We observed a frequent peribronchial locationof lung tumors in O20 mice whereas only 1 OcB-9 tumor was located at abronchus.

We show evidence for genetic basis of 2 robust lung tumor phenotypes:presence of infiltrating lymphocytes and peribronchial location of tumors.The tumor location in O20 and OcB-9 mice could be a consequence of thedifferentiation stage of the lung during transplacental treatment. Lungdevelopment is initiated with migration of foregut endoderm-derived epi-thelial cells into the surrounding splanchnic mesoderm [30]. The mainaspects of lung development are formation of the bronchial tree andalveolar spaces, maturation of the alveolar epithelium, and induction of


surfactant production. Most and perhaps all mouse lung tumors arederived from cells of the alveolar epithelium of the type II cell lineage,although some tumors had been considered to originate from Clara cells[20, 3135].

Transplacental induction of lung tumors in offspring is affected by theday of gestation at carcinogen treatment. Mouse lung tumor numbersincrease following ENU treatment between days 12 and 16 and thendecline through the rest of gestation. The mice in this study were treatedwith ENU on day 18 of gestation. At this age there is a distinct change fromairway development (pseudoglandular period) to extensive branching ofthe distal epithelium during the canalicular stage (days 16.6 to 17.4),resulting in formation of the terminal sacs lined with epithelial cells inte-grating with the mesoderm-derived vasculature. At day 18 of gestation theterminal sac stage (days 17.5 to postnatal day 5) of lung development ischaracterized by a coordinated increase in terminal sac formation and vas-culogenesis and the differentiation of alveolar epithelial type I and II cells[36]. This may affect the number of cells susceptible to carcinogen action,specifically, the cuboidal, fetal alveolar type II precursor cell [33, 3740]and their location with respect to bronchial tree, if the rate of maturationis not the same in the strains tested.

The differentiation of the lung epithelium is known to be regulated byglucocorticoids. Oomen and colleagues [41] showed that glucocorticoid-induced development of alveolar lung volume is influenced by the majorhistocompatibility complex of the mouse (H2) and that glucocorticoidtreatment of fetuses before the administration of carcinogen influencesprenatal ENU induction of lung and intestinal tumors. We hypothesizetherefore that as the differentiation of epithelium proceeds, its propensityto develop tumors changes along the general direction from bronchiolestowards periphery of the lung. The differential location of lung tumors withrespect to the bronchial tree may therefore reflect the degree of differen-tiation of the lung epithelium along the bronchiolesperiphery gradientin the 2 strainsand hence may provide a useful tool to study the relation-ship between target cell differentiation and susceptibility to tumorigenesisby specific pathways. In accordance with this concept, Haigis and collea-gues [42] have shown that the location of intestinal adenomas in Apcmutant heterozygous mice is related to the mechanism of inactivation ofthe wild-type allele. Mapping and identification of the genes controllingthe location of lung tumors in the 2 strains may provide a useful handleto understand the basis of this phenomenon.

Tumor cells produce cytokines that recruit a heterogeneous populationof leucocytes, including lymphocytes [43]. Infiltrating lymphocytes maycontribute to inflammatory reactions that may support or suppress tumorgrowth [43]. Some tumor cells not only regulate their chemokine


expression to help recruitment of inflammatory cells by chemokines butalso increase the local production of lymphokines to stimulate furthergrowth and progression. Indeed, in the OcB strains the presence of infil-trating lymphocytes is strongly associated with presence of regions withhigher degree of cellular dedifferentiation, nuclear atypia, and tumor het-erogeneity [26]. The more frequent presence of infiltrating lymphocytes inO20 tumors may be a contributing factor to their larger size and a moreadvanced stage of progression (M. Hou and P. Demant, unpublished obser-vations). Recently we have shown that genes controlling macrophage func-tions [44] and lymphocyte activation [45] are linked to and possiblyidentical to lung tumor susceptibility genes. Migration of lymphocytesto tumors is dependent on multiple molecules regulating cell adhesion[46, 47] and it is important for the success of antitumor vaccination. Pre-sently we are characterizing the subsets and types of infiltrating lympho-cytes. Identification of genes responsible for this strain difference andelucidation of their function would further elucidate the mechanisms reg-ulating their presence of lymphocytes in tumors and their modifying effectson tumor cells.

In conclusion, we present evidence for genetic basis of 2 robust lungtumor phenotypes: presence of infiltrating lymphocytes and peribronchiallocation of tumors. Their future genetic and molecular analysis will provideadditional markers for a more precise characterization of individual tumorsthat may help to predict their future progression and responses to therapyor vaccination.

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