The Effects of Indirubin Derivative E804 on Inflammatory Profiles in Glioblastoma Multiforme Cell Lines and Subsequent Crosstalk Effect on Tumor Associated THP-1 Macrophages

Glioblastoma Multiforme (GBM) is a highly invasive brain tumor that affects approximately 18,000 people annually in the USA alone yet remains without curative treatment. Median survival with treatment is 14 months, with most treatments only prolonging life for several months while causing severe adverse side effects. There is a need for new therapeutic modalities. Herein I explore two small molecules that show promise in modulating inflammatory signaling in an in vitro GBM model. Indirubins E804 (indirubin-3’-(2,3 dihydroxypropyl)-oximether) and 7BIO (7-Bromoindirubin-3’-oxime) are synthetic derivatives of natural indirubin. Natural indirubin is a bisindole alkaloid derived from tryptophan precursors and is an agonist for the ligand-activated transcription factor aryl hydrocarbon receptor (AHR). Indirubin has been shown to disrupt important cancer-driving signaling such as JAK/STAT3, GSK3, and CDKs. To determine AHR pathway significance in my results, I added a selective AHR antagonist, 6,2’,4’-trimethoxyflavone (TMF), in addition to indirubins. Herein I show that E804 modulates a large array of inflammatory genes, including down-regulation of important autocrine GBM signaling molecules like IL-6 and VEGF, which help drive tumor development. 7BIO, but not E804, increased the expression of STAT3, suggesting that this compound is not suitable for glioma therapy. Additional studies showed that E804 does not fully activate the unfolded protein response (UPR). The UPR is an ER stress pathway that can induce indiscriminate apoptosis - an undesirable side effect in an area as sensitive as the brain. Finally, using transcriptomics combined with pathway analysis, I examined the effects that E804- or E804+TMF-treated glioblastoma cells have on differentiated THP-1 macrophages to model the effects that gliomas may have on glioma-associated macrophages. Glioma-associated macrophages can comprise up to 30% of GBM mass, and, if activated by certain glioma-derived signals, have been implicated in tumor promotion. I found that treated-GBM supernatants inhibited gene profiles in THP-1 macrophages that are linked to tumor progression, such as the Wnt, TLR, and Cell Cycle pathways; enriched gene ontology (GO) terms in up-regulated gene lists involving vascularization and hormone metabolism; and modulated expression of genes associated with macrophage polarization. Taken together, the results of this study demonstrate that E804 and E804 + TMF modulate signaling in GBM cells, with the ability to influence macrophages. These treatments hold promise as an alternative to current modalities using very toxic nitrosourea compounds, and future work should continue to investigate crosstalk between macrophages and GBM, both in vitro and in vivo

Geographic variation in the calling songs and genetics of Bartram's round-winged katydid Amblycorypha bartrami (Tettigoniidae, Phaneropterinae) reveal new species

Previous work on Bartram's round-winged katydid, Amblycorypha bartrami Walker, found inconsistencies in song variation across the species' range. Individuals of purported populations of A. bartrami from sandhills across the southeastern US were collected, recorded, and their genes were sequenced to better understand their population structure and evolution. Significant differences in songs, morphology, and genetics were found among populations from Alabama (AL), Georgia (GA), North Carolina (NC), and South Carolina (SC), and they differed from those of individuals collected from the type locality in Florida (FL). Males from all populations produced songs composed of a series of similar syllables, but they differed in the rates at which syllables were produced as a function of temperature. At temperatures of 25°C, the calling songs of males from populations in northern AL and GA were found to have the highest syllable rates, those from SC had the lowest rates, and those from NC were found to produce songs with doublet syllables at rates that were intermediate between those of males from FL and those of AL and GA. These song differences formed the basis for cluster analyses and principal component analyses, which showed significant clustering and differences in song spectra and morphology among the song morphs. A Bayesian multi-locus, multi-species coalescent analysis found significant divergences from a panmictic population for the song morphs. Populations from GA and AL are closely related to those of A. bartrami in FL, whereas populations from NC and SC are closely related to each other and differ from the other three. Large river systems may have been important in isolating these populations of flightless katydids. Based on the results of our analyses of songs, morphology, and genetics, three new species of round-winged katydids from the southeastern coastal plain and piedmont are described

Supplementary material 1 from: Forrest TG, Scobie M, Brueckner O, Bintz B, Spooner JD (2023) Geographic variation in the calling songs and genetics of Bartram's round-winged katydid Amblycorypha bartrami (Tettigoniidae, Phaneropterinae) reveal new species. Journal of Orthoptera Research 32(2): 153-170. https://doi.org/10.3897/jor.32.96295

Supplementary material 1 from: Forrest TG, Scobie M, Brueckner O, Bintz B, Spooner JD (2023) Geographic variation in the calling songs and genetics of Bartram's round-winged katydid Amblycorypha bartrami (Tettigoniidae, Phaneropterinae) reveal new species. Journal of Orthoptera Research 32(2): 153-170. https://doi.org/10.3897/jor.32.9629

Supplementary material 4 from: Forrest TG, Scobie M, Brueckner O, Bintz B, Spooner JD (2023) Geographic variation in the calling songs and genetics of Bartram's round-winged katydid Amblycorypha bartrami (Tettigoniidae, Phaneropterinae) reveal new species. Journal of Orthoptera Research 32(2): 153-170. https://doi.org/10.3897/jor.32.96295

Supplementary material 4 from: Forrest TG, Scobie M, Brueckner O, Bintz B, Spooner JD (2023) Geographic variation in the calling songs and genetics of Bartram's round-winged katydid Amblycorypha bartrami (Tettigoniidae, Phaneropterinae) reveal new species. Journal of Orthoptera Research 32(2): 153-170. https://doi.org/10.3897/jor.32.9629

Supplementary material 3 from: Forrest TG, Scobie M, Brueckner O, Bintz B, Spooner JD (2023) Geographic variation in the calling songs and genetics of Bartram's round-winged katydid Amblycorypha bartrami (Tettigoniidae, Phaneropterinae) reveal new species. Journal of Orthoptera Research 32(2): 153-170. https://doi.org/10.3897/jor.32.96295

Supplementary material 3 from: Forrest TG, Scobie M, Brueckner O, Bintz B, Spooner JD (2023) Geographic variation in the calling songs and genetics of Bartram's round-winged katydid Amblycorypha bartrami (Tettigoniidae, Phaneropterinae) reveal new species. Journal of Orthoptera Research 32(2): 153-170. https://doi.org/10.3897/jor.32.9629

Supplementary material 2 from: Forrest TG, Scobie M, Brueckner O, Bintz B, Spooner JD (2023) Geographic variation in the calling songs and genetics of Bartram's round-winged katydid Amblycorypha bartrami (Tettigoniidae, Phaneropterinae) reveal new species. Journal of Orthoptera Research 32(2): 153-170. https://doi.org/10.3897/jor.32.96295

Supplementary material 2 from: Forrest TG, Scobie M, Brueckner O, Bintz B, Spooner JD (2023) Geographic variation in the calling songs and genetics of Bartram's round-winged katydid Amblycorypha bartrami (Tettigoniidae, Phaneropterinae) reveal new species. Journal of Orthoptera Research 32(2): 153-170. https://doi.org/10.3897/jor.32.9629