(176b) A Single-Cell Perspective On Signaling and Heterogeneity Maintenance in Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is the most common and lethal brain cancer in adults. The current standard-of-care involves surgical resection followed by administration of radiotherapy and temozolomide. This treatment, however, has generally proven ineffective in patients, with a median 2-year survival rate of about 26.5%. This is largely because this ‘blanket’ therapy fails to account for the complex inter-tumoral and intra-tumoral genomic and functional heterogeneity of the cancer, as well as its myriad interactions with the tumor microenvironment.

    Inter-tumoral heterogeneity in GBM is often associated with the 2 GBM progression pathways: the de novo and progressive pathways. Although the tumors that arise via these separate pathways are histologically indistinguishable, closer inspection reveals that they harbor distinct combinations of genetic aberrations, with each disrupting different players in classic oncogenic and tumor suppressor pathways. Intra-tumoral heterogeneity, on the other hand,involves the development of distinct cellular subcompartments within GBMs that demonstrate different biological behavior, most importantly with respect to drug resistance. A classic example of this involves heterogeneity at the EGFR locus. Studies have indicated that around 50% of all GBMs have amplified levels of wtEGFR and of these, about half harbor the constitutively-active EGFRvIII mutant variant. It has been shown that EGFRvIII+ cells can enhance the proliferation of wtEGFR+ cells via IL-6 paracrine signaling. maximizing overall tumorigenicity. GBM cells are also able to co-opt their stromal compartment to their advantage via the secretion of various immunomodulatory, hypoxic and angiogenic factors. With an occurrence rate of roughly 7 in 100,000 individuals per year, it is clear that targeted therapies that are better able to handle these intricacies have to be devised to combat this disease. This will require a more intimate knowledge of the relevant intercellular pathways that lead to heightened tumorigenicity, supported by advancements in medical genetics that continue to make genomic and transcriptomic screening more economically viable. This will allow us to correlate genomics with function, allowing the design of better treatments for GBM.

    This presentation will focus on elucidating links between the secretomes of distinct cell subpopulations within GBM primary samples and their mutational status at the EGFR locus, as well as their wider genomic profile. To investigate the link between genomics and function, we have used arrays of subnanoliter wells (nanowells) to study primary GBM samples with single-cell resolution. Cells are loaded onto the nanowell device and are allowed to settle via gravity before being stained with various surface markers (e.g., CD45 and EGFR), allowing the discrimination of distinct cell subpopulations via image-based cytometry. Once loaded, the process of ‘microengraving’ is performed to determine the cytokine secretion profiles of the cell(s) in each well: this process involves sealing a glass slide functionalized with appropriate capture antibodies against the loaded nanowell device for a set duration, allowing for the capture of cytokines that can later be detected using fluorescent dye-conjugated detection antibodies. The image-based cytometry and microengraving data are analyzed in an integrated fashion to identify cells of interest. These cells are isolated using a micromanipulator robot into microtiter plates for downstream sequencing, yielding genomic/transcriptomic data linked to the functional data obtained via microengraving.

    Proof-of-concept experiments using the commercially available T98G cell line demonstrate heterogeneity in the secretion of the cytokine IL-6, with 11% of the events registering secretion over four hours. Examination of both single-cell events and multi-cell events suggests secretion occurs in an independent manner—that is, there is no synergy or antagonism of IL-6 secretion on the numbers of IL-6-secreting cells. This example illustrates how the discrete co-cultures enabled in the nanowells provides a unique experimental platform for investigating functional intercellular interactions.  Experiments will also be reported for primary GBM-derived neurosphere lines in microtiter plates to investigate the effects of depletion of various exogenous factors (e.g. EGF) and drug treatments  (e.g. erlotinib) on their bulk functional and genomic profiles, as well as their proliferative capacity and lineage differentiation. This systematic analysis may shed light on possible escape pathways exploited by GBMs to circumvent treatments and promote tumor relapse, as well as revealing critical players in the homeostatic maintenance of heterogeneity. Similar studies recapitulated with single-cell resolution may further reveal possible heterogeneities in response. These functional investigations, coupled with characterization of genetic heterogeneity, should begin to reveal how intra-tumoral diversity promotes survival and maintenance, and may reveal new targets for therapy.