Distinctions Between the Metabolic Changes in Glioblastoma Cells and Glioma Stem-like Cells Following Irradiation

Abstract

Local tumor ablation can be achieved by physical energy-based therapies, such as, irradiation, ultrasound, microwave and radiofrequency. Although tumor antigens released from ablated tumors could induce an anti-tumoral immunity, antigen presentation from ablated tumors are not efficient in inducing a strong systemic immunity. Recently, we demonstrated that immunopriming by LOFU, followed by hypofractionated radiation, reversed immunological tolerance of T cells in draining lymph nodes, induced systemic anti-tumoral immunity, and produced local, regional and metastatic control of murine melanoma (J. Immunol. 196(4):1964-1976). LOFU uses low energy (w500 W/cm 2) and operating frequency of 1 MHz to induce membrane perturbation and mild hyperthermia at the focal zone, without ablating the tissue. Here, we investigate the effect of LOFU on gene expression and cell surface immunomodulation of murine tumor cells. Materials/Methods: LOFU parameters were set at 3-5W of total acoustic output power, 50% duty cycle, 1.5 seconds, and 1 mm spacing (TIPS, Philips Research). After LOFU treatment of a cell pellet, cells were cultured for various time points for RNA sequencing, qPCR, immunofluorescence and flow cytometry studies. Tumor cell lines included human DU145 prostate cancer cells and mouse RM1 and TPSA23 prostate cancer, Lewis lung carcinoma (3LL) and breast cancer (4T1). TPSA23 was derived from TRAMPC1 which was genetically engineered to express human PSA. Statistical evaluation distinguished gene expression changes by 1.5-fold, with false detection rate (FDR) <0.05. Wilcoxon rank sum was used to detect statistical significance in gene expression from RT-PCR results (p<0.05). Results: Gene expression studies (RNAseq and qRT-PCR) demonstrate that LOFU (3W) induced key genes involved in the unfolded protein response (UPR) pathway. Compared to untreated DU145 cells, LOFU induced ERN1 three-folds, HSPA1B and HSPH1 by 8.79 folds and HSP105/110 by 6.18-fold within 6-24 hrs. In separate experiments, there was a significant increase in Hspa1 RNA expression (132-fold) and CHOP expression (2.5 fold) 24 h post-LOFU, in RM-1 and 4T1 cells, respectively. Flow cytometry and immunofluorescence in 3LL, 4T1 and RM1 cells demonstrated the translocation of stress proteins, BiP, calreticulin and HSP70 to the cell surface. There was induction of cell surface death receptors (CD40 and Fas), immune checkpoint inhibitors (PD-L1 and PD-L2) and class I MHC in 3LL, 4T1 and TPSA23 cell lines. Conclusion: These studies demonstrate that LOFU induces expression of genes involved in UPR and endoplasmic stress response. Translocation of stress chaperones to the cell surface along with increased expression of MHC I and death receptors contribute to the immunomodulatory properties of LOFU.

The Molecular and Cellular Strategies of Glioblastoma and Non-Small-Cell Lung Cancer Cells Conferring Radioresistance

“…GSCs have been reported to express cell surface CSCs markers, such as stage-specific mouse embryonic antigen, CD34, CD44, α6- integrin, CD133, L1CAM (L1 Cell Adhesion Molecule), CD54, and A2B5 [ 97 , 217 , 227 , 228 , 229 , 230 , 231 , 232 , 233 , 234 , 235 ]; cytoskeleton proteins (also referred as; intermediate filaments or nanofilaments), such as glial fibrillary acidic protein (GFAP) [ 236 ], vimentin [ 237 , 238 , 239 ], and Nestin; transcription factors, such as SOX2, Nanog, Oct3/4 [ 240 , 241 ], nuclear factor erythroid 2-related factor 2 (Nrf2) [ 241 , 242 ], oligodendrocyte transcription factor (Olig2) [ 243 ], FoxM1 [ 244 ], and zinc finger protein 281 (ZNF281) [ 245 ], POU class 3 Homeobox 2 [ 246 ], and melanocyte inducing transcription factor (MITF) [ 247 ]; posttranscriptional factors, such as Musashi 1 [ 248 ] and microRNAs [ 249 ]; polycomb (Pc) transcriptional suppressors, such as enhancer of zeste homolog 2 and BMI 1 [ 248 ]; transcriptional co-activators, such as YAP/TAZ [ 171 ] and TRRAP [ 250 ], yet no single marker is able to define a general GSC population. It has been reported that GSCs metabolism undergoes different changes than that of traditional GBM tumor cells following IR [ 251 ]. Gene expression analyses of GBM cells treated by IR have revealed that many genes are modulated after treatment [ 252 , 253 , 254 ].…”

“…After repairing the cellular damage, the cells may re-enter the cell cycle upon stimulation by specific growth factors, such as E2F and CDK2 [ 463 ]. It has been shown that, after IR, GSCs are more quiescent than GBM cells that express elevated levels of glycolysis and oxidative phosphorylation, the so-called “Warburg effect”, whereas GSCs show metabolic signs of quiescence, such as a diminished non-essential amino-acid synthesis, and unchanged levels of glycolytic and oxidative metabolites [ 251 ]. Earlier studies have demonstrated that Ras-related C3 botulinum toxin substrate 2 induces aberrant proliferation of quiescent NSCLC after IR exposure to a single dose of 2 Gy via promoting JUN-B expression through megakaryoblastic leukemia 1—serum response factor (MAL-SRF) pathway [ 464 ].…”

Radiation-Induced Alterations in the Recurrent Glioblastoma Microenvironment: Therapeutic Implications

“…It has been said that what doesn't kill you makes you stronger, and in certain respects GBM exemplifies such adaptive behavior. Tumors adapt to radiation-induced oxidative stress through several mechanisms, including metabolic shifts ( 63 ), elevated antioxidant peptide production, and intra-tumoral hypoxia generation ( 150 , 151 ).…”