A preclinical orthotopic model for glioblastoma recapitulates key features of human tumors and demonstrates sensitivity to a combination of MEK and PI3K pathway inhibitors

Current therapies for glioblastoma multiforme (GBM), the highest grade malignant brain tumor, are mostly ineffective, and better preclinical model systems are needed to increase the successful translation of drug discovery efforts into the clinic. Previous work describes a genetically engineered mouse (GEM) model that contains perturbations in the most frequently dysregulated networks in GBM (driven by RB, KRAS and/or PI3K signaling and PTEN) that induce development of Grade IV astrocytoma with properties of the human disease. Here, we developed and characterized an orthotopic mouse model derived from the GEM that retains the features of the GEM model in an immunocompetent background; however, this model is also tractable and efficient for preclinical evaluation of candidate therapeutic regimens. Orthotopic brain tumors are highly proliferative, invasive and vascular, and express histology markers characteristic of human GBM. Primary tumor cells were examined for sensitivity to chemotherapeutics and targeted drugs. PI3K and MAPK pathway inhibitors, when used as single agents, inhibited cell proliferation but did not result in significant apoptosis. However, in combination, these inhibitors resulted in a substantial increase in cell death. Moreover, these findings translated into the in vivo orthotopic model: PI3K or MAPK inhibitor treatment regimens resulted in incomplete pathway suppression and feedback loops, whereas dual treatment delayed tumor growth through increased apoptosis and decreased tumor cell proliferation. Analysis of downstream pathway components revealed a cooperative effect on target downregulation. These concordant results, together with the morphologic similarities to the human GBM disease characteristics of the model, validate it as a new platform for the evaluation of GBM treatment

FIGURE 4 from Functional Heterogeneity in MET Pathway Activation in PDX Models of Osimertinib-resistant EGFR-driven Lung Cancer

Heterogeneity in MET polysomy and MET amplification in osimertinib-resistant EGFR-mutant NSCLC. A, Representative phospho-MET IHC (several are reused from Fig. 2D), MET FISH images and MET FISH scoring from PDXs with MET polysomy. B, Representative phospho-MET IHC, MET FISH images and MET FISH scoring from longitudinally collected tumor samples from patient LAT006 at first progression on osimertinib, second progression after osimertinib rechallenge, and upon further progression on chemoimmunotherapy. Scale bars, 50 µm.</p

FIGURE 3 from Functional Heterogeneity in MET Pathway Activation in PDX Models of Osimertinib-resistant EGFR-driven Lung Cancer

Osimertinib and savolitinib combination suppresses AKT signaling in MET amplified osimertinib-resistant EGFR-mutant NSCLC PDXs. EGFR and MET pathway protein expression in MET amplified (A) and MET polysomy (B) LAT006 PDXs treated with either vehicle, osimertinib, savolitinib, and osimertinib plus savolitinib. Bar graphs show the relative quantification of phosphorylated proteins normalized to total protein expression in MET amplified (C) and MET polysomy (D) LAT006 PDXs.</p

Supplementary Table from Functional Heterogeneity in MET Pathway Activation in PDX Models of Osimertinib-resistant EGFR-driven Lung Cancer

Supplementary Table provides clinical, molecular and passaging details for the PDXs generated in this study</p

FIGURE 1 from Functional Heterogeneity in MET Pathway Activation in PDX Models of Osimertinib-resistant EGFR-driven Lung Cancer

Generation of spatial and temporally heterogenous osimertinib-resistant EGFR-mutant NSCLC PDX models and treatment study design. A, Schematic diagram of the prospective clinical trial of LAT for osimertinib-treated EGFR-mutant lung cancer (RT: radiotherapy). PDXs were generated from osimertinib-resistant tumor tissue either at first or second progression on osimertinib or after standard of care (S.O.C) therapy. B, Multi-region and temporal tumor samples from surgical resections or biopsies used for PDX generation are shown for each individual patient. Putative mechanism of resistance to osimertinib as evidenced by exome and or transcriptome sequencing (Roper et al., Cell Reports Medicine 2020) is shown below each set of PDXs. Color denotes timing of sample acquisition. Red: first progression on osimertinib; Green: second progression on osimertinib; Blue: progression on S.O.C treatment. C, Illustrations of PDX generation from 3 patients with EGFR-mutant lung cancer with MET polysomy by FISH (MET ≥ 4.0 and MET/CEP7 ratio is MET amplification by FISH (MET/CEP7 ratio ≥2.0 or ≥6 MET copies per cell) as a mechanism of resistance to osimertinib. D, Study design for treatment with MET inhibitor (savolitinib) with a third-generation EGFR TKI (osimertinib). PDXs with spatial heterogeneity in MET pathway activation (LAT001_6B and LAT001_9B), PDXs with temporal heterogeneity in MET pathway activation (LAT006_2B and LAT006_0118) and an additional validation PDX (LAT015_6B) were treated with vehicle, osimertinib, savolitinib, and osimertinib plus savolitinib combination followed by assessment of efficacy and identification of predictive markers.</p

FIGURE 5 from Functional Heterogeneity in MET Pathway Activation in PDX Models of Osimertinib-resistant EGFR-driven Lung Cancer

Phospho-MET expression is an indicator of MET activity post-osimertinib treatment; and proposed clinical flow diagram for treating EGFR-mutant NSCLC with evidence of MET pathway activation after osimertinib resistance. Representative phospho-MET IHC, MET FISH images and MET FISH scoring from pre- and post-osimertinib resistant tumors from patient LAT028 (multiple spatially heterogenous post-osimertinib resistant tumors shown; A) and from patient LAT021 (B). Scale bars, 50 µm. C, Clinical flow diagram for osimertinib-resistant EGFR-mutant NSCLC with evidence of MET pathway activation.</p

FIGURE 2 from Functional Heterogeneity in MET Pathway Activation in PDX Models of Osimertinib-resistant EGFR-driven Lung Cancer

Efficacy and determinants of response to osimertinib and savolitinib combination among osimertinib-resistant EGFR-mutant NSCLC PDX models with spatially and temporally heterogenous MET pathway activation. Tumor growth inhibition studies in MET polysomy (A) and MET amplified (B) PDXs. Dashed vertical lines delineate when treatment was stopped. Asterisks signify statistical significance between osimertinib and savolitinib combination and osimertinib alone treatment arms. P values were calculated by t test. P values C, Association between response to osimertinib and savolitinib combination and IHC features (phospho-MET, c-MET), copy number and FISH parameters (number of MET copies and MET/CEP7 ratio). Response is defined as >25 days until reaching tumor size endpoint in osimertinib and savolitinib combination compared with osimertinib treatment alone. Individual circles represent a unique tumor for each represented PDX model. D, Representative c-MET and phospho-MET IHC images of PDX tumors with and without response to osimertinib and savolitinib combination. Scale bars, 50 µm.</p

“Glowing Head” Mice: A Genetic Tool Enabling Reliable Preclinical Image-Based Evaluation of Cancers in Immunocompetent Allografts

<div><p>Preclinical therapeutic assessment currently relies on the growth response of established human cell lines xenografted into immunocompromised mice, a strategy that is generally not predictive of clinical outcomes. Immunocompetent genetically engineered mouse (GEM)-derived tumor allograft models offer highly tractable preclinical alternatives and facilitate analysis of clinically promising immunomodulatory agents. Imageable reporters are essential for accurately tracking tumor growth and response, particularly for metastases. Unfortunately, reporters such as luciferase and GFP are foreign antigens in immunocompetent mice, potentially hindering tumor growth and confounding therapeutic responses. Here we assessed the value of reporter-tolerized GEMs as allograft recipients by targeting minimal expression of a luciferase-GFP fusion reporter to the anterior pituitary gland (dubbed the “Glowing Head” or GH mouse). The luciferase-GFP reporter expressed in tumor cells induced adverse immune responses in wildtype mouse, but not in GH mouse, as transplantation hosts. The antigenicity of optical reporters resulted in a decrease in both the growth and metastatic potential of the labeled tumor in wildtype mice as compared to the GH mice. Moreover, reporter expression can also alter the tumor response to chemotherapy or targeted therapy in a context-dependent manner. Thus the GH mice and experimental approaches vetted herein provide concept validation and a strategy for effective, reproducible preclinical evaluation of growth and response kinetics for traceable tumors.</p></div

Generation of the rGH-ffLuc-eGFP (“Glowing Head”) genetically engineered mouse.

<p><b>A</b>, Structure of the expression vector for generation of Glowing Head (GH) transgenic mice. Expression of a firefly luciferase-eGFP fusion gene (ffLuc-eGFP) was targeted to the mouse anterior pituitary gland by using the rat growth hormone promoter (rGH) and human growth hormone gene sequences, which include a polyadenylylation site (hGHpA)²⁰. <b>B</b>, Optical expression pattern of transgene in GH mice as visualized by BL imaging. Reporter activity was detected in the anterior pituitary gland of both genders and the testes of male mice. <b>C</b>, Serum levels of growth hormone from age-matched GH mice and wildtype (WT) c-Brd mice was assessed by ELISA (mean ± SE). Blood was withdrawn at the same time of day. No significant differences in circulating growth hormone levels between the GH and WT mice were found. <b>D</b>, ffLuc-eGFP-labeled LLC tumors were subcutaneously transplanted into WT, GH, and NOD-SCID mice. Blood was withdrawn to prepare sera when tumors reached 500 mm³, and the serum levels of anti-GFP antibody were analyzed by ELISA. The levels of anti-GFP antibody in WT mice are significantly higher than those in GH and NOD-SCID mice (p<0.005), but no difference was found between those in GH and NOD-SCID mice (p = 0.19). The sera from healthy mice without tumor transplantation served as controls to define zero point.</p