Citation: Moretti, I.F.; Lerario, A.M.; Sola, P.R.; Macedo-da-Silva, J.; Baptista, M.d.S.; Palmisano, G.; Oba-Shinjo, S.M.; Marie, S.K.N. GBM Cells Exhibit Susceptibility to Metformin Treatment According to TLR4 Pathway Activation and Metabolic and Antioxidant Status. Cancers 2023, 15, 587. https://doi.org/10.3390/ cancers15030587 Academic Editor: Edward Pan Received: 20 December 2022 Revised: 10 January 2023 Accepted: 14 January 2023 Published: 18 January 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cancers Article GBM Cells Exhibit Susceptibility to Metformin Treatment According to TLR4 Pathway Activation and Metabolic and Antioxidant Status Isabele Fattori Moretti 1, * , Antonio Marcondes Lerario 2 , Paula Rodrigues Sola 1 , Janaína Macedo-da-Silva 3 , Mauricio da Silva Baptista 4 , Giuseppe Palmisano 3 , Sueli Mieko Oba-Shinjo 1 and Suely Kazue Nagahashi Marie 1, * 1 Laboratory of Molecular and Cellular Biology (LIM 15), Department of Neurology, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo 01246-903, SP, Brazil 2 Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes, University of Michigan, Ann Arbor, MI 48108, USA 3 GlycoProteomics Laboratory, Department of Parasitology, ICB, University of Sao Paulo, São Paulo 05508-000, SP, Brazil 4 Biochemistry Department, Institute of Chemistry, Universidade de Sao Paulo, São Paulo 05508-900, SP, Brazil * Correspondence: imortetti@usp.br (I.F.M.); sknmarie@usp.br (S.K.N.M.); Tel.: +55-11-3061-7458 (S.K.N.M.) Simple Summary: An analysis of metformin (MET) treatment in combination with temozolomide (TMZ) in two glioblastoma cell lines, U87MG and A172, stimulated with lipopolysaccharide (LPS), a TLR4 agonist was conducted. Both cells presented blunted mitochondrial respiration leading to oxidative stress after MET treatment, and decreased cell viability after MET + TMZ treatment. U87MG cells presented increased apoptosis after MET + LPS + TMZ treatment by increment of ER stress, and downregulation of BLC2. A172, with an upregulated antioxidant background, including SOD1, exhibited cell cycle arrest after MET + TMZ treatment. The observed differential response was associated with a distinct metabolic status: glycolytic/plurimetabolic (GPM) subtype in U87MG and mitochondrial (MTC) in A172. TCGA-GBM-RNASeq in silico analysis showed that GPM-GBM cases with an activated TLR4 pathway might respond to MET, but the concomitant CXCL8/IL8 upregulation may demand a combination treatment with an IL8 inhibitor. MET combined with an antioxidant inhibitor, such as anti-SOD1, may be indicated for MTC-GBM cases. Abstract: Glioblastoma (GBM) is an aggressive brain cancer associated with poor overall survival. The metabolic status and tumor microenvironment of GBM cells have been targeted to improve ther- apeutic strategies. TLR4 is an important innate immune receptor capable of recognizing pathogens and danger-associated molecules. We have previously demonstrated the presence of TLR4 in GBM tumors and the decreased viability of the GBM tumor cell line after lipopolysaccharide (LPS) (TLR4 agonist) stimulation. In the present study, metformin (MET) treatment, used in combination with temozolomide (TMZ) in two GBM cell lines (U87MG and A172) and stimulated with LPS was ana- lyzed. MET is a drug widely used for the treatment of diabetes and has been repurposed for cancer treatment owing to its anti-proliferative and anti-inflammatory actions. The aim of the study was to investigate MET and LPS treatment in two GBM cell lines with different metabolic statuses. MET treatment led to mitochondrial respiration blunting and oxidative stress with superoxide production in both cell lines, more markedly in U87MG cells. Decreased cell viability after MET + TMZ and MET + LPS + TMZ treatment was observed in both cell lines. U87MG cells exhibited apoptosis after MET + LPS + TMZ treatment, promoting increased ER stress, unfolded protein response, and BLC2 downregulation. LPS stimulation of U87MG cells led to upregulation of SOD2 and genes related to the TLR4 signaling pathway, including IL1B and CXCL8. A172 cells attained upregulated antioxidant gene expression, particularly SOD1, TXN and PRDX1-5, while MET treatment led to cell-cycle arrest. In silico analysis of the TCGA-GBM-RNASeq dataset indicated that the glycolytic plurimetabolic (GPM)-GBM subtype had a transcriptomic profile which overlapped with U87MG cells, suggesting Cancers 2023, 15, 587. https://doi.org/10.3390/cancers15030587 https://www.mdpi.com/journal/cancers