Pre-clinical imaging for establishment and comparison of orthotopic non-small cell lung carcinoma: in search for models reflecting clinical scenarios.

OBJECTIVE:: Clinically relevant animal models of non-small cell lung carcinoma (NSCLC) are required for the validation of novel treatments. We compared two different orthotopic transplantation techniques as well as imaging modalities to identify suitable mouse models mimicking clinical scenarios. METHODS:: We used three genomically diverse NSCLC cell lines [National Cancer Institute (NCI)-H1703 adenosquamous cell carcinoma, NCI-H23 adenocarcinoma and A549 adenocarcinoma) for implanting tumour cells either as spheroids or cell suspension into lung parenchyma. Bioluminescence imaging (BLI) and contrast-enhanced cone beam CT (CBCT) were performed twice weekly to monitor tumour growth. Tumour histological data and microenvironmental parameters were determined. RESULTS:: Tumour development after spheroid-based transplantation differs probably due to the integrity of spheroids, as H1703 developed single localised nodules, whereas H23 showed diffuse metastatic spread starting early after transplantation. A549 transplantation as cell suspension with the help of a stereotactic system was associated with initial single localised tumour growth and eventual metastatic spread. Imaging techniques were successfully applied to monitor longitudinal tumour growth: BLI revealed highly sensitive qualitative data, whereas CBCT was associated with less sensitive quantitative data. Histology revealed significant model-dependent heterogeneity in proliferation, hypoxia, perfusion and necrosis. CONCLUSION:: Our developed orthotopic NSCLC tumours have similarity with biological growth behaviour comparable to that seen in the clinic and could therefore be used as attractive models to study tumour biology and evaluate new therapeutic strategies. The use of human cancer cell lines facilitates testing of different genomic tumour profiles that may affect treatment outcomes. ADVANCES IN KNOWLEDGE:: The combination of different imaging modalities to identify tumour growth with subsequent use in treatment planning and orthotopic transplantation techniques to develop initially single lesions to ultimate metastases pave the way towards representative pre-clinical NSCLC models for experimental testing of novel therapeutic options in future studies.

An orthotopic model of lung cancer to analyze primary and metastatic NSCLC growth in integrin alpha1-null mice.

The role of matrix metalloproteinase (MMP)9 in lung cancer progression is controversial. MMP9 promotes local tumor progression and distant metastasis in mouse models by enhancing extracellular matrix degradation, releasing VEGF from extracellular matrix and promoting vascular pericyte recruitment. Furthermore, increased plasma MMP9 expression levels in human subjects with metastatic non-small cell lung cancer (NSCLC) inversely correlates with survival. In contrast, MMP9 can benefit the host by generating inhibitors of endothelial cell proliferation such as angiostatin and NC1 domains of collagen IV. To better understand the role of host MMP9 on the primary growth and metastatic potential of NSCLC, we performed an orthotopic model of NSLC in integrin alpha1-null mice (a genetic model for increased MMP9). In these mice we observed decreased number, size and vascularization of primary NSCLC tumors when compared to wild type controls. In addition, decreased number and size of NSCLC-derived metastases were evident in the alpha1-null mice. Furthermore, pharmacological inhibition of MMPs in the alpha1-null mice at the time of tumor cell injection resulted in an increase in the number of both primary and metastatic lung cancer as compared to untreated mice, suggesting that primary growth and metastases of NSCLC are worsened by the early inhibition of MMPs. In conclusion, although MMP9 may potentially promote tumor growth and metastasis, production of MMP-dependent anti-angiogenic factors seems to override these effects and protects the host from NSCL growth and progression.

Combination treatment with erlotinib and pertuzumab against human tumor xenografts is superior to monotherapy.

In many solid tumors, overexpression of human epidermal growth factor receptors (e.g., HER1/EGFR and HER2) correlates with poor prognosis. Erlotinib (Tarceva) is a potent HER1/EGFR tyrosine kinase inhibitor. Pertuzumab (Omnitarg), a novel HER2-specific, recombinant, humanized monoclonal antibody, prevents heterodimerization of HER2 with other HERs. Both mechanisms disrupt signaling pathways, resulting in tumor growth inhibition. We evaluated whether inhibition of both mechanisms is superior to monotherapy in tumor cell lines expressing different HER levels. Human non-small cell lung cancer (NSCLC) cells (Calu-3: HER1/EGFR 0+, HER2 3+; QG56: HER1/EGFR 2-3+, HER2 0+) and breast cancer cells (KPL-4: HER1/EGFR 2-3+, HER2 3+) were implanted into BALB/c nu/nu mice and severe combined immunodeficient beige mice, respectively. Tumor-bearing mice (n = 12 or 15 per group) were treated with vehicle (Captisol or buffer), erlotinib (orally, 50 mg/kg/d), pertuzumab (i.p. 6 mg/kg/wk with a 2-fold loading dose), or erlotinib and pertuzumab for 20 (QG56), 27 (KPL-4), or 49 (Calu-3) days. Drug monotherapy had antitumor activity in all models. Tumor volume treatment-to-control ratios (TCR) with erlotinib were 0.36 (Calu-3), 0.79 (QG56), and 0.51 (KPL-4). Pertuzumab TCR values were 0.42, 0.51, and 0.64 in Calu-3, QG56, and KPL-4 models, respectively. Combination treatment resulted in additive (QG56: TCR 0.39; KPL-4: TCR 0.38) or greater than additive (Calu-3: TCR 0.12) antitumor activity. Serum tumor markers for NSCLC (Cyfra 21.1) and breast cancer (soluble HER2) were markedly inhibited by combination treatment (80-97% in Calu-3 and QG56; 92% in KPL-4), correlating with decreased tumor volume. Overall, erlotinib and pertuzumab are active against various human xenograft models, independently of HER1/EGFR or HER2 expression. A combination of these HER-targeted agents resulted in additive or greater than additive antitumor activity.

NNK-induced hamster lung adenocarcinomas over-express beta2-adrenergic and EGFR signaling pathways.

Pulmonary adenocarcinoma (PAC) is the most common type of human lung cancer. A diagnosis of PAC, history of non-smoking and presence of mutations in the EGFR are predictive factors for responsiveness of lung cancer to EGFR-specific tyrosine kinase inhibitors. Unfortunately, less than 50% of PAC cases demonstrate this mutation-based responsiveness. Our immunohistochemical analysis of NNK-induced PAC in hamsters demonstrates the simultaneous over-expression of a beta2-adrenergic receptor pathway, including PKA, cAMP, CREB and phosphorylated CREB and of an EGFR pathway, including over-expression of EGFR-specific phosphorylated tyrosine kinase, Raf-1 and ERK1/2 and their phosphorylated forms. These findings implicate, for the first time, PKA/CREB-mediated signaling in the development and regulation of any type of lung cancer. In light of reports that NNK acts as a beta-adrenergic agonist and that beta-blockers inhibit the growth of PAC of Clara cell lineage in the NNK hamster model and in human cancer cell lines from smokers, our current data suggest transactivation of the EGFR pathway via beta-adrenergic signaling as a novel regulatory mechanism in a subpopulation of PACs in smokers. Taken together, these data point to PKA/CREB as novel targets for the development of cancer therapeutics for PAC patients non-responsive to EGFR-specific tyrosine kinase inhibitors.

Pulmonary neuroendocrine carcinoma in a four-month-old dog.

A 4-month-old male Siberian Husky dog had a history of coughing, high fever and anorexia. Thoracic radiographs revealed increased radiographic density in the cranial and middle lobes of the right lung, with pleural effusion. Cytological examination of the pleural fluid suggested carcinomatous pleuritis. Right-side thoracotomy and resection of the cranial and middle lobes were performed. Histopathological examination of the resected tissue revealed an anaplastic large cell carcinoma. The tumour cells were positive for neuron specific enolase and also contained neuroendocrine granules. A particularly unusual feature of this case of pulmonary neuroendocrine carcinoma was the young age of the affected animal.

K-ras activation in non-small cell lung cancer in the dog.

To investigate the role of K-ras mutations in canine non-small cell lung cancer, we first determined the nucleotide sequence of the normal canine K-ras gene and then examined 21 canine lung tumors for activating K-ras mutations. Canine K-ras was analyzed by direct sequencing of polymerase chain reaction products generated with oligonucleotide primers derived from the human K-ras sequence. Four nucleotide differences were found between the canine and human K-ras sequence from position 5 to 211. The deduced amino acid sequence of the canine gene was identical to that of the human. Activated K-ras alleles were detected in 5 of the 21 canine lung tumors examined. The activating lesions were point mutations, predominantly in codon 12. Of the 14 adenocarcinomas examined, 2 (14%) had K-ras mutations. Two of 5 (40%) adenosquamous carcinomas and the only large cell carcinoma also contained activated alleles. The overall frequency of K-ras point mutation in non-small cell lung cancer (25%) is similar to that reported in human non-small cell lung cancer. We conclude that K-ras activation by point mutation is associated with, but not necessary for, non-small cell lung cancer development in the dog.