Novel technique for preclinical assessment of targeted therapies to inhibit both primary and metastatic non-small cell lung cancer

Author: Gunisha Arora, Medical and Scientific Writer, Scientific Development
Date: February, 2024
 

Lung cancer remains a formidable challenge in healthcare as the leading cause of cancer-related fatalities. Constituting 80% of all lung cancer cases, non-small cell lung cancer (NSCLC) is of particular concern due to the development of brain metastasis observed in 20%-40% of NSCLC patients, contributing to poor prognosis, recurrence and a post-metastasis survival time of only one to two months if left untreated.¹ 

Current therapeutic modalities, including surgery, radiotherapy, chemotherapy and immunotherapy, inadequately address the underlying biological and molecular mechanisms of brain metastasis in NSCLC, necessitating the identification of targeted therapies to combat primary NSCLC while effectively inhibiting metastatic progression. With epidermal growth factor receptor (EGFR)  mutation being one of the high-risk factors for brain metastasis of NSCLC with stage IIIB/IV,¹ a third-generation tyrosine kinase inhibitor (TKI), Osimertinib was identified and approved as a preferred first-line treatment for EGFR-mutated NSCLC patients, including patients with brain metastasis.² A recent study indicates that combining Osimertinib with chemotherapy in patients with NSCLC and brain metastasis leads to improved clinical outcomes.³

To facilitate the comprehensive evaluation of metastatic brain disease in the presence of primary tumor, dual-disease preclinical models are essential. To address this unmet need, Labcorp Discovery Oncology has developed and optimized xenograft models for dual-disease evaluation using bioluminescence imaging (BLI) for two human NSCLC cell lines: NCI-H1975-Luc and PC-9-Luc-mCh-Puro. As previously described in “NCI-H1975-Luc and PC-9-Luc: two models for evaluating brain metastases from non-small cell lung cancer”, both cell lines harbor unique mutational EGFR T790M status and grow reliably and reproducibly. In this spotlight, we examine whether the xenograft models for metastatic brain disease via a direct intracranial (IC) implant coupled with a subcutaneous (SC) primary tumor effectively allow evaluation of the response to treatment at both locations. 

In separate studies, each cell line was implanted subcutaneously and intracranially into female nude mice. Once tumors were established at both implant sites, oral treatment was initiated with Osimertinib 25 mg/kg QD for 14 days. SC tumor progression was measured by digital calipers and IC tumor burden was monitored by BLI using an IVIS® Spectrum (Perkin Elmer). Both SC primary and IC metastatic tumors were harvested after the third treatment and analyzed for total and phospho-EGFR (pEGFR) and total and phospho-ERK1/2(pERK1/2) using the AlphaLISA™ SureFire Ultra Detection (Perkin Elmer) system.

Response to Treatment

In the NCI-H1975-Luc model, treatment with Osimertinib at 25 mg/kg orally for 14 days was highly efficacious at both tumor locations resulting in delay of metastatic brain disease and regression of the primary SC tumor. There were several mice that presented clinical signs and body weight loss indicative of progressive IC tumor growth. The primary tumors had much higher levels of pEGFR and pERK1/2 than the metastatic brain tumors, which substantially decreased across all three time points following treatment with Osimertinib. At the metastatic site, the expression of pEGFR and pERK1/2 was slightly reduced in the face of Osimertinib treatment, suggesting that the treatment was able to pass through the blood-brain barrier and reach the tumor. 

Figure 1. NCI-H1975-Luc tumor growth kinetics with response to Osimertinib treatment for both SC and IC implants. Bar below X-axis indicates treatment duration.

NCI-H1975-Luc Primary (SC Implanted) Tumors: AlphaLISA Analysis 

NCI-H1975-Luc Metastatic (IC Implanted) Tumors: AlphaLISA Analysis 

Figure 2: AlphaLISA for total and phospho EGFR and ERK1/2 from both SC and IC NCI-H1975-Luc tumors.

In the PC-9-Luc-mCh-Puro model, Osimertinib treatment at 25 mg/kg orally for 14 days was highly effective at both tumor locations resulting in stable metastatic disease in the brain and primary SC tumor regression. The primary tumors had much higher levels of pEGFR than the metastatic brain tumors; pEGFR expression was highly suppressed and the pERK1/2 levels were only slightly inhibited regardless of the time points following Osimertinib treatment. At the metastatic site, the expression of pEGFR and pERK1/2 were relatively unchanged even though there was an inhibition of tumor growth based on the longitudinal BLI analysis. Data from both models suggest that bioavailability in the brain is decreased compared to the SC implanted primary tumor.

Figure 3. PC-9-Luc-mCh-Puro tumor growth kinetics with response to Osimertinib treatment for both SC and IC implants. Bar below X-axis indicates treatment duration.

PC-9-Luc-mCh-Puro Primary (SC Implanted) Tumors: AlphaLISA Analysis 

PC-9-Luc-mCh-Puro Metastatic (IC Implanted) Tumors: AlphaLISA Analysis 

Figure 4: AlphaLISA for total and phospho EGFR and ERK1/2 from both SC and IC PC-9-Luc-mCh-Puro tumors

This innovative approach aims to bridge existing gaps in preclinical oncology models, offering a promising avenue for refining treatment strategies and improving outcomes in patients with NSCLC and brain metastasis. 

Contact us to learn more about how our subcutaneous, metastatic and orthotopic models coupled with our ex vivo analyses services can be used to advance your preclinical research.