Characterization of a murine lung adenocarcinoma (LAC1), a useful experimental model to study progression of lung cancer

Journal of Experimental Therapeutics and Oncology Vol. 0 2011 1

Characterization of a murine lung adenocarcinoma (lac1), a useful experimental model to study progression of lung cancer

Mariana Piegari1,2, Susana Ortiz3, María del Pilar Díaz4, Aldo R Eynard1,2,5, Mirta A. Valentich1,2,5

1Instituto de Biología Celular, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, CP5000, Ciudad Universitaria, Córdoba, Argentina2Cátedra de Biología Celular, Histología y Embriología, Universidad Nacional de Córdoba, Ciudad Universitaria, Córdoba, CP5000, Argentina3Asignatura Patología Humana, Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Hospital Nacional de Clínicas, Córdoba, Argentina4Cátedra de Bioestadística, Escuela de Nutrición, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba. Ciudad Universitaria. Córdoba. Argentina5Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina

*Correspondence to: Mirta A. Valentich, Instituto de Biología Celular, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, CP5000, Ciudad Universitaria, Córdoba, Argentina. Telephone: 54-51-4334020. E-mail: [email protected]

(Received April 8, 2011; revised June 14, 2011; accepted July 9, 2011)

Lung cancer is one of the most important avoidable causes of death around the world, the most widespread carcinoma, with a very poor prognosis, and is the leading cause of cancer death in both developed and developing countries. We report morphological and biological behavior characteristics of a tumor that arose in only one BALB/c mouse of an experimental group treated with urethane, a chemical lung-tumorigenic agent. Morphological and immunochemical analysis indicated phenotypic com-patibility with a lung adenocarcinoma. The tumor was named LAC1 (lung adenocarcinoma 1). Implant success in eight LAC1-bearing mice generations was 100%, with a fast evolution (58 survival days) and good metastatic capacity (41% of animals with metastases). The tumor induced a paraneoplastic syndrome characterized by anemia, neutrophilia, cachexia, splenomegaly and thymic atrophy. The lymphoproliferation to Con A was altered in tumor-bearing mice. This lung adenocarcinoma may be a useful experimental model for studying tumor pro-gression, paraneoplastic syndromes and immunology in carcinogenic studies.

Key words: lung adenocarcinoma, mice, histopathology, immunohistopathology, lymphoproliferation

IntroductIon

Lung cancer is the leading cause of cancer-related death worldwide (1). Despite increasing understand-

ing of the biology of tumors, there have been no major advances in the therapy of this type of cancer, and the need to develop better diagnostic techniques and thera-pies is urgent (1,2).

There have been a considerable number of gene-en-gineering experimental studies, but there are only a few animal models to investigate lung cancer, like Lewis lung carcinoma (LLC) that was isolated and cloned from metastatic lung nodules of C57BL/6 mice, and LP07 lung adenocarcinoma that appeared spontane-ously in Balb/c mice (2-5).

Animal models are a useful tool to increase under-standing of many aspects of the carcinogenesis process and allow for the testing of novel chemopreventives, therapeutics and screening methods. Also, they could be used to approach to paraneoplastic syndromes knowl-edge in order to improve quality of life of cancer patients (1,6-10).

Spontaneous occurrence of lung tumors in experi-mental animals, and especially rodents, is extremely rare (2). However, these may be studied after induction through physical and chemical carcinogens, in mod-els modified by gene-engineering or in experimental tumor-transplanted animal models (6-8,10-12).

In our laboratory, studying the modulation by die-tary lipids of tumor growth patterns of lung adenomas in urethane-treated mice (7), only one animal developed neck lymph node metastases. When small pieces from this metastasis were implanted subcutaneously in mice,

Journal of Experimental Therapeutics and Oncology, Vol. 0, pp. 00–00 © 2011 Old City Publishing, Inc.Reprints available directly from the publisher Published by license under the OCP Science imprint,Photocopying permitted by license only a member of the Old City Publishing Group.

M. Piegari et al.

2 Journal of Experimental Therapeutics and Oncology Vol. 0 2011

100% tumor transplant success was obtained, which led us to consider the feasibility of using this as a trans-plantable tumor model. The purpose of this study was thus to develop an experimental model of a transplant-able alveolar carcinoma of the lung with its histopatho-logical, electron microscopy, immunocytochemical and biological behavior characterization.

MaterIals and Methods

Animals

Two-three month old inbred BALB/c mice of both sexes were employed. Standard pellet diet (GEPSA feeds, Animal Nutrition Division, Córdoba, Argentina) and water were provided ad libitum. Mice were inspected daily and body weight was recorded once a week.

Procedures involving animals were approved by the Commission of the SECYT-FCM-UNC.

Tumor

The original tumor tissue was excised from a metas-tasis in the lymph node of neck developed in a single mouse from a urethane-treated experimental group with lung adenomas (7).

A total of 148 animals were used. The tumor was propagated in 8 successive animal generations in a total number of 122 mice. The animals received a tro-car subcutaneous implant (1mm3) of tumor in 0.2 ml of PBS into the right lateral flank, under light anesthesia as usual in our laboratory (8). 26 mice (without tumor implant) were used as controls for spleen and thymus weight and lymphoproliferation. Tumor diameter was recorded once a week.

Animals were autopsied when they died spontane-ously or were sacrificed in advance because of their poor physical condition as is usual practice in our labo-ratory (8). Only recently dead animals were autopsied, in order to avoid unreliable sources of data points.

Histopathology

Tumor tissue and any other organs with macroscopic abnormality were routinely processed for histopatho-logic evaluation through light microscopy.

Electron microscopy

Pieces of tumor tissue were fixed in 2% glutaralde-hyde, post-fixed with 2% osmium tetroxide, embedded

in epoxy resin, sectioned and stained with uranyl ace-tate and lead citrate, for observation under transmission electron microscope.

CK7, TTF-1 and S100 protein expression

Immunohistochemistry was used to detect S100 (DAKO, polyclonal), cytokeratin 7 (CK7) (DAKO, mon-oclonal OV-TL 12/30) and thyroid transcription factor 1 (TTF-1) (DAKO, monoclonal) protein expressions in tumor and lung metastases.

Tumor parameters

Tumor parameters were measured in eight genera-tions of tumor bearing animals. The parameters recorded were: Successful transplantation (ST): the percentage of successful implants; Tumor latency time (TLT): the period from transplant to the palpable detection of the tumor; Post-transplantation survival time (PST): the period of time from tumor transplant until spontaneous death, or sacrifice when showing severe loss of weight and marked physical distress; Net survival time (NST): the period of time from palpable detection of the tumor until spontaneous death or sacrifice.

Additionally, during necropsy, presence and location of metastases were recorded (8).

Spleen and thymus changes

At autopsy, spleen and thymus were weighed and spleen processed for histopathologic evaluation through light microscopy.

Hematological parameters

Blood studies were performed by hematocrit and erythrocyte and leukocyte count in control and tumor-bearing mice.

Spleen lymphocytes proliferation

Spleens of 10 tumor-bearing mice and 5 control mice were used. Tumor-bearing mice were sacrificed after one month of tumor transplant and controls at age-equivalent sacrifice.

Spleens were removed aseptically, homogenized, placed in RPMI 1640 medium and treated with red blood cell lysing buffer (Hybri-Max – SIGMA-Aldrich). Mononuclear cells obtained were cultured in a 96-well plate at 2×105 viable cells in 100 µl RPMI

Development of an experimental transplantable lung tumor model


1640 medium with L-glutamine and without phenol red (SIGMA-Aldrich), supplemented with 13% fetal bovine serum (FBS) and 0.1% gentamycin. Cells were stimulated with 2 or 5 µg/ml of Concanavalin A, during 72 hours at 37ºC and 5% CO

2 and cell viability meas-

ured with MTT assay.

MTT assay

Cell viability was measured using MTT assay modified by Graber and Losa (13). Cultured cells were added with 30 µl of MTT (Sigma-Aldrich) solution (2.5 mg/ml in RPMI-1640 without phenol red) to each well. After 4 hs of incubation at 37ºC and 5% CO

2, the

converted dye was solubilized with acidic isopropa-nol (0.04 N HCl-isopropyl-alcohol). Absorbance was measured at 560 nm with background subtraction at 630-690 nm.

Statistical analysis

The significance of the differences in the assays was adjusted to a generalized lineal model with a Binomial or Poisson-type stochastic component, with logistic-type connection function and one-dimensional linear predictor. The model was adjusted with the GLIM4 (2009) program for any discrete variable. The signifi-cance was valued at p ≤0.05.

To determine parameter associations, logistic regres-sion was used.

results

Macroscopic appearance

The tumor was formed of a subcutaneous voluminous mass, with hard consistency and moderately defined limits and occasionally with epidermal ulcerate lesions. Tumor sections showed a whitish-yellowish color with foci of hemorrhage and necrosis and some cystic formations.

Histology

The microscopic appearance of the tumor subcu-taneous primary lesion and lung metastases was of a moderately differentiated adenocarcinoma. The tumor propagated was named LAC1 (lung adenocarcinoma 1) and the number of passage was clarified in brackets.

The primary tumor, propagated tumors and pulmo-nary metastases in all of the transplanted animal gen-erations showed quite uniform epithelial neoplastic cells with macronucleus, round to oval nuclei with conspicu-ous nucleoli. The cells were mingled in solid clusters and duct-like structures covered by an uncharacteristic cubic epithelium sometimes with free rounded cells inside. A lot of mitosis, some atypical, was observed (Fig. 1).

Additionally, the primary tumor as well as the tumor and the metastases of first tumoral generations of trans-planted animals showed keratin presence in the form of typical “keratin pearls”; disappearing completely since the fourth generation.

Figure 1. Histological features of LAC1 tumor. H-E. 200X. Inside: details of glandular pattern 400X.

M. Piegari et al.


Tumor immunohistological features

Tumor cells were immunoreactive to intracytoplas-mic S100 and CK 7 proteins and to intranuclear TTF1 protein expression (Fig. 2).

Electron microscopy

LAC1 tumor cells showed large irregular nuclei with indentations. Mitochondria, rough endoplasmic reticu-lum, Golgi apparatus, free ribosomes and frequent dense and multivesicular bodies were observed in the cytoplasm. The cellular membrane surface was gener-ally irregular with microvilli. Desmosomes were seen between tumor cells (Fig. 3).

Tumor diagnosis

Histology, electron microscopy and immunohisto-chemical studies defined the tumor as a lung adenocar-cinoma, which was named LAC1.

Tumor parameters

None of the measured tumor parameters showed sig-nificant differences between animals of different sexes.

Successful transplantation (ST): This was 100% in all transplanted animals.

Tumor Latency time (LT), Post-transplantation sur-vival time (PST) and Net survival time (NST) showed some differences between generations of tumor-bearing

Figure 2. Immunohistochemical features of LAC1 tumor. A- S100 intracytoplasmic expression. 400X. B- CK 7 intracytoplasmic expression. 100X. C- TTF1 intranuclear expression. 400X. Lower left inside shows the intranuclear positivity of normal lung cells. 100X.

Figure 3. Electronmicroscopic appearance of LAC1 tumor cells. Notice big nuclei (N), prominent Golgi complex (G), multivesicular bodies (M), dense bodies (D) and desmosomes (arrows).



animals. Taking all generations together, the average LT was 12.81 days, PST was 58.09 days and NST 45.58 days (Table).

Curve of tumor growth, constructed with diameters measured during tumor development, showed a gradual increase, reaching greatest diameter observed (100%) on until day 35 after subcutaneous implant. Before this, tumor growth was stabilized until spontaneous death or sacrifice (Fig. 4).

Animal weight (AW): Animals of all generations suf-fered a marked loss of corporal weight (roughly 23%) measured since the tumor implant until the autopsy, after tumor removal. This fact added to anorexia and to a notable loss of muscle and fat mass (data not shown), have suggested a state of cachexia, one of the more common paraneoplastic syndromes (14,15).

Tumor diameter and tumor weight did not reach signif-icant differences between generations (data not shown).

Presence of metastases

41% of LAC1-bearing mice showed macroscopic lung metastases significantly associated (p<0.0002) with post-transplantation survival time, without differ-ences between generations.

Splenomegaly

LAC1 tumor-bearing mice autopsy showed a signifi-cantly enlarged spleen (p< 0.0389). It was friable and exhibited microscopic structural alterations presenting

remains of lymphoid follicles, intense hemorrhages with blood collection forming organized hematomas, with a homogeneous pink–color compatible with fibrin mate-rial, frequently associated with numerous neutrophil (polymorphonuclear leukocyte) accumulations. The splenic capsule was separated from the parenchyma, leaving spaces that were sometimes occupied by inflam-matory elements, especially neutrophils. Trabecular remains were observed as residual stroma.

Statistical analysis of spleen weight showed sig-nificantly (p< 0.0389) marked splenomegaly in LAC1-bearing mice of all animal generations compared with control animal spleens. Spleen weight was at least 25% greater than that of controls (Fig. 5) and 78% of tumor-bearing mice showed this, without significant differ-ences between generations.

Thymic atrophy

Thymus weight of LAC1-bearing mice of all animal generations showed significant reduction (p< 0.026), being at least 35% lighter than controls (Fig 6), and this was characteristic in 97% of tumor-bearing mice. In addition, a more than 97% reduction in thymus weight was seen in 72% of LAC1-bearers. Results were similar in all generations.

Hematological parameters

LAC1-bearing mice showed significantly dimin-ished erythrocyte number (p< 0.0013) and hematocrit

Figure 4. Tumor growth. Around 35 days after subcutaneous implant, tumor reached the greatest diameter (100%), stabilizing growth until mice spontaneous death or sacrifice.

M. Piegari et al.


(p< 0.0103) value and increased leukocyte number (p< 0.0005) due to neutrophilia (Fig. 7).

Spleen lymphoproliferation changes

Different concentrations of Con A (2 or 5 µg/ml) induced similar lymphoproliferation levels in con-trol mice but were unable to induce proliferation in tumor-bearing mice mononuclear cells. Basal viability of LAC1-bearing mice mononuclear cells was signifi-cantly higher than in control mice (Fig. 8). There were no differences in cell viability between Con A-treated cultures from control and tumor-bearing mice (Fig. 8).

Tumor maintenance

Pieces of tumor tissue were successfully maintained with 10% DMSO as cryoprotectant in culture medium with 45% FBS, allowing a high transplant outcome (90%) when defrosted.

Figure 5. Spleen weight in control and LAC1-bearing mice. Splenomegaly was observed in 78% of tumor-bearing animals. Data are expressed as mean ± SEM.

Figure 6. Thymus weight in control and LAC1 bearing mice. Thymic atrophy was observed in 97% of tumor-bearing animals. Data are expressed as mean ± SEM.

Figure 7. Changes in peripheral blood counts. Data are expressed as mean ± SEM.

dIscussIon

A murine lung alveolar carcinoma (LAC1) was established as an experimental model. It could be main-tained frozen, was highly successful when transplanted after defrosting and showed clinical and biological sim-ilarities to certain human lung cancers.

Histopathology studies showed characteristics that were phenotypically compatible with lung adenocar-cinoma. Immunohistochemical characterization of the tumor cells showed intracytoplasmic S100 protein and CK7 and intranuclear TTF1 positive immunoreactions. S100 proteins are commonly up-regulated in tumors and this is often associated with tumor progression (16,17). Members of the S100 family, such as S100A11 protein, may be an important regulatory factor in promoting the invasion and metastasis of non-small cell lung cancer (18). On the other hand, it has been reported that posi-tive staining of CK7 is expressed in 97%–100% (19), and of TTF-1 in 70%–90% of primary lung adenocarci-nomas (20). Both immunohistochemical markers, CK7



and TTF-1, have been useful for the diagnosis of lung tumors (21).

Optical and electron microscopy studies indicated a high cellular undifferentiation corresponding with the high malignancy observed in the LAC1 tumor (22).

The biological characterization through eight gener-ations of LAC1-bearing mice showed that its transplant had an effectiveness of 100%, together with a fast evo-lution (58 days) and a good capacity to produce metas-tases (41% of animals with metastases).

LAC1 tumor induced a paraneoplastic syndrome char-acterised by anemia, neutrophilia, cachexia, splenomeg-aly and thymic atrophy (2,15,23). It has been shown that paraneoplastic syndromes are not produced directly by the tumor or its metastases but by secreting factors pro-duced by tumor cells or by normal cells in response to the tumor (24). Further studies must be performed in order to determine the presence of secreting factors produced by the LAC1 tumor.

Cachexia was observed in all LAC1-bearing mice; this condition has been reported to be associated with abnormalities including anorexia, weight loss (prefer-ential loss of somatic muscle and fat mass), and anemia (14). LAC1 could be used to measure other metabolic alterations characteristic of cachexia, like altered hepatic glucose and lipid metabolism.

Anemia is commonly observed in lung cancer and is a prevalent condition associated with cancer in approxi-mately 40-64% of patients (25,26). The correction of anemia has been showed to impact the quality of life and survival of cancer patients (26).

The elevated neutrophilia observed in LAC1-bearing mice is one of the most common paraneoplastic syn-dromes associated with cancer (2,27). Because the

leukocytosis is usually associated with the presence of colony-stimulating factors (G-CSF and GM-CSF) and furthermore, for the reason that it has been noted that 40% of the CSF-producing tumors are lung carcinomas (2,28), LAC1 could be used to investigate this topic.

The splenomegaly observed was due to the replace-ment of normal tissue by blood collections forming organized hematomas, and fibrin areas associated with significant accumulation of neutrophil polymorphonu-clear leukocytes with a loss of normal spleen architecture, concomitantly with hematological alterations. In fact, spleen hyperplasia has been associated with hematopoi-etic alterations such as marked leukocytosis and expan-sion of intrasplenic neutrophil granulocytes (2,29).

The profound thymic involution observed during the growth of the LAC1 could be associated with a severe depletion of the most abundant subset of thymocytes, CD4+8+ immature cells as it was shown by Lopez et al. (30) in mammary tumorigenesis of mice. Also, the tumor could induce a failure in peripheral immuno-surveillance as stated by Mandal et al. (23) in Ehrlich ascites carcinoma-bearing mice. Thymus weight reduc-tion was probably secondary to the cachexia because it is known that this was significantly affected in malnu-trition state (31,32).

Loss of proliferative response to Con A of mononu-clear spleen cells of tumor-bearing mice must be due to in vivo anergy induction from tumor presence (33-35), while an increase in basal viability with respect to con-trol mice might indicate in vitro cell proliferation by tumor factor prestimulation affecting Con A mitogenic response.

The development of murine models of lung cancer may aid in our understanding of lung tumor biology and

Figure 8. Changes in lymphoproliferation. There were significant differences in basal viability and lymphoproliferation between controls and LAC1-bearing mice. Data are expressed as mean ± SEM.

M. Piegari et al.


facilitate the development and testing of novel thera-peutic approaches and methods for early diagnosis (6). LAC1, a novel experimental tumor, has several proper-ties that make it a promising model for studying tumor progression, paraneoplastic syndromes and tumor immunology and its probable modification by epige-netic nutritional factors. However, this tumor model has rapid development which could be a disadvantage to long term studies.

Since LAC1 is a lung tumor, its potential interest is increased because this is the main cause of death by neoplasia in the world. In addition, it is a model that behaves clinically and biologically in a similar manner to the evolution of the disease in humans.

acknowledgeMents

The authors are grateful for the excellent animal assistance of Mr. Ricardo Mattos. This work was sup-ported by a grant from the Secretaría de Ciencia y Tecnología (SECYT) of the National University of Cór-doba, Argentina.

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Characterization of a murine lung adenocarcinoma (LAC1), a useful experimental model to study progression of lung cancer