The effects of tumor treating fields and temozolomide in MGMT expressing and non-expressing patient-derived glioblastoma cells

AbstractA recent Phase 3 study of newly diagnosed glioblastoma (GBM) demonstrated the addition of tumor treating fields (TTFields) to temozolomide (TMZ) after combined radiation/TMZ significantly increased survival and progression free survival. Preliminary data suggested benefit with both methylated and unmethylated O-6-methylguanine-DNA methyl-transferase (MGMT) promoter status. To date, however, there have been no studies to address the potential interactions of TTFields and TMZ. Thus, the effects of TTFields and TMZ were studied in vitro using patient-derived GBM stem-like cells (GSCs) including MGMT expressing (TMZ resistant: 12.1 and 22GSC) and non-MGMT expressing (TMZ sensitive: 33 and 114GSC) lines. Dose-response curves were constructed using cell proliferation and sphere-forming assays. Results demonstrated a ⩾10-fold increase in TMZ resistance of MGMT-expressing (12.1GSCs: IC50=160μM; 22GSCs: IC50=44μM) compared to MGMT non-expressing (33GSCs: IC50=1.5μM; 114GSCs: IC50=5.2μM) lines. TTFields inhibited 12.1 GSC proliferation at all tested doses (50–500kHz) with an optimal frequency of 200kHz. At 200kHz, TTFields inhibited proliferation and tumor sphere formation of both MGMT GSC subtypes at comparable levels (12.1GSC: 74±2.9% and 38±3.2%, respectively; 22GSC: 61±11% and 38±2.6%, respectively; 33GSC: 56±9.5% and 60±7.1%, respectively; 114 GSC: 79±3.5% and 41±4.3%, respectively). In combination, TTFields (200kHz) and TMZ showed an additive anti-neoplastic effect with equal efficacy for TTFields in both cell types (i.e., ± MGMT expression) with no effect on TMZ resistance. This is the first demonstration of the effects of TTFields on cancer stem cells. The expansion of such studies may have clinical implications

Identification of Mom7, a Novel Modifier of ApcMin/+ on Mouse Chromosome 18

The ApcMin mouse model of colorectal cancer provides a discrete, quantitative measurement of tumor multiplicity, allowing for robust quantitative trait locus analysis. This advantage has previously been used to uncover polymorphic modifiers of the Min phenotype: Mom1, which is partly explained by Pla2g2a; Mom2, a spontaneous mutant modifier; and Mom3, which was discovered in an outbred cross. Here, we describe the localization of a novel modifier, Mom7, to the pericentromeric region of chromosome 18. Mom7 was mapped in crosses involving four inbred strains: C57BL/6J (B6), BTBR/Pas (BTBR), AKR/J (AKR), and A/J. There are at least two distinct alleles of Mom7: the recessive, enhancing BTBR, AKR, and A/J alleles and the dominant, suppressive B6 allele. Homozygosity for the enhancing alleles increases tumor number by approximately threefold in the small intestine on both inbred and F1 backgrounds. Congenic line analysis has narrowed the Mom7 region to within 7.4 Mb of the centromere, 28 Mb proximal to Apc. Analysis of SNP data from various genotyping projects suggests that the region could be as small as 4.4 Mb and that there may be five or more alleles of Mom7 segregating among the many strains of inbred mice. This has implications for experiments involving ApcMin and comparisons between different or mixed genetic backgrounds

Colon Cancer Tumorigenesis Initiated by the H1047R Mutant PI3K.

The phosphoinositide 3-kinase (PI3K) signaling pathway is critical for multiple important cellular functions, and is one of the most commonly altered pathways in human cancers. We previously developed a mouse model in which colon cancers were initiated by a dominant active PI3K p110-p85 fusion protein. In that model, well-differentiated mucinous adenocarcinomas developed within the colon and initiated through a non-canonical mechanism that is not dependent on WNT signaling. To assess the potential relevance of PI3K mutations in human cancers, we sought to determine if one of the common mutations in the human disease could also initiate similar colon cancers. Mice were generated expressing the Pik3caH1047R mutation, the analog of one of three human hotspot mutations in this gene. Mice expressing a constitutively active PI3K, as a result of this mutation, develop invasive adenocarcinomas strikingly similar to invasive adenocarcinomas found in human colon cancers. These tumors form without a polypoid intermediary and also lack nuclear CTNNB1 (β-catenin), indicating a non-canonical mechanism of tumor initiation mediated by the PI3K pathway. These cancers are sensitive to dual PI3K/mTOR inhibition indicating dependence on the PI3K pathway. The tumor tissue remaining after treatment demonstrated reduction in cellular proliferation and inhibition of PI3K signaling

<i>Fc</i>^<i>+</i><i>Pik3ca</i>^{<i>H1047R</i>} colon cancers are similar to those in <i>Fc</i>^<i>+</i> <i>Pik3c</i>a^<i>p110*</i> mice.

<p>In both of these <i>Pik3ca</i> mutant models, deeply invasive cancers are seen with the vast majority of the tumors having penetrated below the muscularis mucosa. This is in contrast to <i>Apc</i>^<i>Min</i> colon tumors, which typically are adenomatous tumors with no or just superficial invasion. Abundant mucin is present within both the <i>Fc</i>^<i>+</i> <i>Pik3ca</i>^{<i>H1047R</i>} and <i>Fc</i>^<i>+</i> <i>Pik3c</i>a^<i>p110*</i> colon cancers. These <i>Pik3ca</i> mutant cancers also demonstrate increased proliferation, as measured by nuclear Ki67, in comparison to <i>Apc</i>^<i>Min</i> tumors. In addition, phosphorylated RPS6 and ERK1/2 are increased above that seen in the <i>Apc</i>^<i>Min</i> colon lesions. Scale bar for low magnification images = 1mm. Enlargements are 10x magnifications of the areas outlined in the low magnification images. Min, <i>Apc</i>^<i>Min</i>.</p

<i>Fc</i>^<i>+</i><i>Pik3ca</i>^{<i>H1047R</i>} colon cancers develop through a non-canonical pathway.

<p>Histological examination demonstrates that these cancers are flat without a polypoid component. Low grade dysplasia (tubular adenoma) has not been identified in these or the <i>Fc</i>^<i>+</i> <i>Pik3c</i>a^<i>p110*</i> mice. At higher magnification, malignant glands above the muscularis mucosa can be identified which appear to be originating from the crypt bases without identification of surface low grade dysplasia. CTNNB1 staining demonstrates that nuclear CTNNB1 is absent in both of the <i>Pik3ca</i> mutant models, but is present in the <i>Apc</i>^<i>Min</i> controls. Scale bar for low magnification image = 1mm. High magnification H&E image is a 6x magnification of the area indicated in the image to the left. Scale bar for CTNNB1 images = 100μm.</p

The PI3K pathway is activated in <i>Apc</i>^<i>Min/+</i>, <i>Fc</i>^<i>+</i> <i>Pik3c</i>a^<i>p110*</i>, and <i>Fc</i>^<i>+</i> <i>Pik3ca</i>^{<i>H1047R</i>} mice.

<p>Immunoblotting demonstrates robust phosphorylation of AKT in <i>Apc</i>^<i>Min</i>, <i>Fc</i>^<i>+</i> <i>Pik3c</i>a^<i>p110*</i> and <i>Fc</i>^<i>+</i> <i>Pik3ca</i>^{<i>H1047R</i>} colon tumors (A). Increased phosphorylation of RPS6 and 4EBP1 beyond that seen in the <i>Apc</i>^<i>Min</i> lesions is observed in the <i>Fc</i>^<i>+</i> <i>Pik3ca</i>^{<i>H1047R</i>} and <i>Fc</i>^<i>+</i> <i>Pik3c</i>a^<i>p110*</i> colon tumors. The more aggressive phenotype seen in the <i>Fc</i>^<i>+</i> <i>Pik3c</i>a^<i>p110*</i> mice, with a greater number and decreased latency, is associated with an increased phosphorylation of 4EBP1 compared to <i>Fc</i>^<i>+</i> <i>Pik3ca</i>^{<i>H1047R</i>} tumors (B).</p

Mutant PI3K can lead to colon cancer development.

<p>Approximately 60% of <i>Fc</i>^<i>+</i> <i>Pik3ca</i>^{<i>H1047R</i>} mice develop tumors within the colon which can result in these mice becoming moribund. At necropsy large colon tumors are found extending through the colonic wall (A). Upon resection, multiple lesions can be identified within the colon and can be over 1 cm in size (A and B). Following histological sectioning, H&E staining demonstrates that these lesions are invasive mucinous adenocarcinomas of the colon without a predominant intra-luminal component (C). Higher magnification demonstrates an abundant desmoplastic reaction with surrounding mucin lakes lined with epithelial cancer cells (D). D size bar = 200 μm.</p

Dual PI3K/mTOR inhibition induces treatment responses in <i>Fc</i>^<i>+</i> <i>Pik3ca</i>^{<i>H1047R</i>} colon cancers.

<p><i>Fc</i>^<i>+</i><i>Pik3ca</i>^{<i>H1047R</i>} mice were treated with NVP-BEZ235 (35mg/kg/day) or control once daily by oral gavage for 14 days. These mice underwent dual hybrid 18F-FDG PET/CT imaging at baseline and then 24 hours following the last dose of study drug (A, arrows denote tumors pre- and post-treatment). A significant reduction in tumor volume, as measured on the CT images, was detected in the NVP-BEZ235 group (p = 0.008; B). In addition, there was a trend for a slight decrease in the median SUV for those cancers treated with the PI3K/mTOR inhibitor. In one control mouse, two tumors were observed on baseline imaging, but not detected on follow-up PET/CT imaging.</p