Chimeric antigen receptor (CAR) T cells represent an exciting therapeutic strategy with improved survival outcomes for patients with hematological malignancies. However, the efficacy of CAR T-cell therapy in the treatment of solid tumors remains suboptimal due to therapeutic barriers associated with the solid tumor microenvironment. We investigated whether ionizing radiation could improve vascular perfusion and CAR T-cell delivery in an EGFRvIII-expressing B16F10 melanoma model. Tumors received radiation doses of 2–12 Gy, and perfusion was evaluated at multiple time points using immunofluorescence detection of intravenously administered fluorescent dyes. We found that a single 8-Gy dose of ionizing radiation produced the most significant increase in B16F10 tumor perfusion 4 h after irradiation. Consistently, the irradiation of tumors 4 h prior to a systemic administration of EGFRvIII-targeting CAR T cells led to higher intratumoral CAR T-cell accumulation than in non-irradiated tumors. This approach also resulted in a significantly delayed tumor growth and improved survival relative to radiation or CAR T cells alone. Interestingly, the CD28ζ EGFRvIII-CAR T-cell levels substantially increased in irradiated tumors over time relative to 4-1BBζ EGFRvIII-CAR T cells and produced greater tumor growth delays and survival improvements in comparison to 4-1BBζ EGFRvIII-CAR T cells administered at a 10-fold higher concentration. Taken together, these data highlight the importance of co-stimulatory domains in CAR T-cell function in vivo and demonstrate that irradiating tumors prior to systemic CAR T-cell infusion can increase CAR T-cell infiltration and efficacy in solid tumors.

Combating resistance to targeted therapy remains a major challenge to improving lung cancer care. Epithelial-mesenchymal transition (EMT) in tumour cells is an established non-genetic resistance mechanism to EGFR tyrosine kinase inhibitors (TKI) that is also associated with worse outcome in patients. Here we demonstrate that integrin-linked kinase (ILK) is an important driver of EMT-mediated TKI resistance in lung adenocarcinoma (LUAD) by promoting a drug-tolerant persister (DTP) cell phenotype. Our results indicate that high ILK expression is associated with EMT in LUAD patients and that genetic suppression of ILK can limit EMT progression and reduce the viability of DTP cells by impairing YAP activation, ultimately improving osimertinib (Osi) sensitivity in LUAD cells. Importantly, LUAD cells with high ILK expression are able to persist during EGFR-TKI treatment, acquiring additional genetic and phenotypic alterations to develop EGFR-TKI resistance. To improve clinical translatability of our findings, we showed that pharmacological inhibition of ILK can suppress EMT and improve Osi response in LUAD cells. Lastly, we found that strong immunohistochemistry staining of ILK in patient biopsies was significantly associated with and may be used to predict receptor tyrosine kinase-independent mechanisms of EGFR-TKI resistance. Overall, our results suggest that ILK is an important regulator of EGFR-TKI response and may be exploited as a predictor for acquired resistance, providing evidence for co-targeting ILK with EGFR to better control minimal residual disease and EGFR-TKI resistance in lung cancer.

Abstract The solid tumor microenvironment is a key determinant of successful cancer therapy, with hypoxic tumor cells known to contribute to poor therapeutic outcome after radiation therapy, chemotherapy, and surgery. Several targeted therapies have been developed against hypoxic tumor cells that have shown promise in combination with radiation therapy in pre-clinical models and early phase clinical trials, although the difficulty inherent in targeting hypoxic cells has limited the overall efficacy of this approach. Our recent data suggest that hypoxia can develop in solid tumors due to the activity of sub-populations of cancer-associated fibroblasts (CAFs), and that therapeutically targeting CAF activation can restrict the development of tumor hypoxia and prime tumors to respond to radiation therapy. Angiotensin II type 1 receptor (AT1R) blockers (ARBs) are anti-hypertensive agents that inhibit the activity of the signaling peptide angiotensin II derived from the renin-angiotensin system. ARBs can also exhibit anti-fibrotic activity by blocking the AT1R expressed on myofibroblasts in various tissues. We recently found that the ARB telmisartan inhibits the deposition of collagen I in solid tumors, increasing net tumor perfusion and stabilizing microregional tumor blood flow. Collagen I can be produced by CAFs, although recent clinical data have identified multiple, functionally-diverse CAF subsets in breast and ovarian tumors. To understand how ARBs affect the solid tumor microenvironment, we wanted to determine how the ARB telmisartan influences the activity of CAF subsets and whether telmisartan-associated changes in perfusion impact hypoxia and radiation response in solid tumors. We identified several murine CAF subsets that could be discriminated by cell surface marker expression across a panel of seven tumor xenograft lines. Using single cell RNAseq of cells isolated from tumors with or without ARB treatment, we found that telmisartan altered the transcriptome of CAF subsets and inhibited CAF activation. In tumors containing myofibroblast-like CAFs, telmisartan decreased collagen deposition and eliminated ‘acute’ hypoxia that can be caused by unstable microregional perfusion. Telmisartan-treated tumors also had improved response to ionizing radiation therapy. Taken together, our data indicate that CAFs contribute to tumor hypoxia and radiation resistance of solid tumors in an AT1R-dependent manner. Using a retrospective cohort of over 1,100 oropharyngeal cancer patients, we also found that patients taking ARBs for hypertension had dramatically improved therapeutic outcomes after radiation therapy. Our work indicates that CAFs contribute to the development of hypoxia in solid tumors and suggests that angiotensin receptor blockers represent a novel targeted therapy to inhibit CAF activity, modify the solid tumor microenvironment, reduce tumor hypoxia, and improve radiation response. Citation Format: Che-Min Lee, Brennan J. Wadsworth, Rachel A. Cederberg, Zi Han Li, Kiersten N. Thomas, Meredith Clark, Rocky Shi, Michael Hall, Nikita Telkar, Ryan Urban, Wan L. Lam, Sarah N. Hamilton, Kevin L. Bennewith. Targeting the angiotensin receptor in cancer-associated fibroblasts improves tumour perfusion, oxygenation, and radiotherapy response. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr B016.

Background Radiation therapy is commonly used to treat head and neck cancer patients, and response may be improved by combining radiation with preexisting medications. Based on recent preclinical and retrospective patient data, we hypothesized that antihypertensive drugs may improve radiotherapy outcomes.Methods Retrospective analyses were conducted on 1077 oropharyngeal squamous cell carcinoma and 608 nasopharyngeal carcinoma patients, all of whom received radiation therapy. Univariate and multivariate analyses were conducted to assess overall survival, disease-specific survival, and locoregional control for cancer patients taking angiotensin receptor blockers (ARBs), angiotensin-converting enzyme inhibitors, calcium channel blockers, or beta blockers compared with propensity score-matched groups of patients not taking these medications.Results Oropharyngeal squamous cell carcinoma patients taking antihypertensive medications were statistically older and had higher Charlson Comorbidity Indices at diagnosis. However, these patients had statistically significant improved overall survival, disease-specific survival, and locoregional control compared with propensity score-matched oropharyngeal squamous cell carcinoma patients who were not taking antihypertensive medications, with ARB users showing the greatest improvements. Antihypertensive drugs did not affect outcomes in the nasopharyngeal carcinoma patient cohort.Conclusion The use of antihypertensive medications, and particularly ARBs, was associated with improved outcomes in oropharyngeal squamous cell carcinoma patients who had more advanced age and higher Charlson Comorbidity Indices at diagnosis. This study supports future prospective testing of ARBs in conjunction with radiation therapy in this group of higher risk oropharyngeal squamous cell carcinoma patients. Additionally, this study illustrates the need to use propensity score matching to identify patient subgroups that may benefit from a given treatment in retrospective analyses.

BackgroundLung cancer is the leading cause of cancer related death worldwide, mainly due to the late stage of disease at the time of diagnosis. Non-invasive biomarkers are needed to supplement existing screening methods to enable earlier detection and increased patient survival. This is critical to EGFR-driven lung adenocarcinoma as it commonly occurs in individuals who have never smoked and do not qualify for current screening protocols.MethodsIn this study, we performed mass spectrometry analysis of the secretome of cultured lung cells representing different stages of mutant EGFR driven transformation, from normal to fully malignant. Identified secreted proteins specific to the malignant state were validated using orthogonal methods and their clinical activity assessed in lung adenocarcinoma patient cohorts.ResultsWe quantified 1020 secreted proteins, which were compared for differential expression between stages of transformation. We validated differentially expressed proteins at the transcriptional level in clinical tumor specimens, association with patient survival, and absolute concentration to yield three biomarker candidates: MDK, GDF15, and SPINT2. These candidates were validated using ELISA and increased levels were associated with poor patient survival specifically in EGFR mutant lung adenocarcinoma patients.ConclusionsOur study provides insight into changes in secreted proteins during EGFR driven lung adenocarcinoma transformation that may play a role in the processes that promote tumor progression. The specific candidates identified can harnessed for biomarker use to identify high risk individuals for early detection screening programs and disease management for this molecular subgroup of lung adenocarcinoma patients.

Mediator, a co-regulator complex required for RNA Polymerase II activity, interacts with tissue-specific transcription factors to regulate development and maintain homeostasis. We observe reduced Mediator subunit MED15 expression in endocrine hormone-producing pancreatic islets isolated from people living with type 2 diabetes and sought to understand how MED15 and Mediator control gene expression programs important for the function of insulin-producing β-cells. Here we show that Med15 is expressed during mouse β-cell development and maturation. Knockout of Med15 in mouse β-cells causes defects in β-cell maturation without affecting β-cell mass or insulin expression. ChIP-seq and co-immunoprecipitation analyses found that Med15 binds β-cell transcription factors Nkx6-1 and NeuroD1 to regulate key β-cell maturation genes. In support of a conserved role during human development, human embryonic stem cell-derived β-like cells, genetically engineered to express high levels of MED15, express increased levels of maturation markers. We provide evidence of a conserved role for Mediator in β-cell maturation and demonstrate an additional layer of control that tunes β-cell transcription factor function.

BACKGROUND:Identification of driver mutations and development of targeted therapies has considerably improved outcomes for lung cancer patients. However, significant limitations remain with the lack of identified drivers in a large subset of patients. Here, we aimed to assess the genomic landscape of lung adenocarcinomas (LUADs) from individuals without a history of tobacco use to reveal new genetic drivers of lung cancer. METHODS:Integrative genomic analyses combining whole-exome sequencing, copy number, and mutational information for 83 LUAD tumors was performed and validated using external datasets to identify genetic variants with a predicted functional consequence and assess association with clinical outcomes. LUAD cell lines with alteration of identified candidates were used to functionally characterize tumor suppressive potential using a conditional expression system both in vitro and in vivo. RESULTS:We identified 21 genes with evidence of positive selection, including 12 novel candidates that have yet to be characterized in LUAD. In particular, SNF2 Histone Linker PHD RING Helicase (SHPRH) was identified due to its frequency of biallelic disruption and location within the familial susceptibility locus on chromosome arm 6q. We found that low SHPRH mRNA expression is associated with poor survival outcomes in LUAD patients. Furthermore, we showed that re-expression of SHPRH in LUAD cell lines with inactivating alterations for SHPRH reduces their in vitro colony formation and tumor burden in vivo. Finally, we explored the biological pathways associated SHPRH inactivation and found an association with the tolerance of LUAD cells to DNA damage. CONCLUSIONS:These data suggest that SHPRH is a tumor suppressor gene in LUAD, whereby its expression is associated with more favorable patient outcomes, reduced tumor and mutational burden, and may serve as a predictor of response to DNA damage. Thus, further exploration into the role of SHPRH in LUAD development may make it a valuable biomarker for predicting LUAD risk and prognosis.

Organotypic cultures of murine brain slices are well-established tools in neuroscience research, including electrophysiology studies, modeling neurodegeneration, and cancer research. Here, we present an optimized ex vivo brain slice invasion assay that models glioblastoma multiforme (GBM) cell invasion into organotypic brain slices. Using this model, human GBM spheroids can be implanted with precision onto murine brain slices and cultured ex vivo to allow tumour cell invasion into the brain tissue. Traditional top-down confocal microscopy allows for imaging of GBM cell migration along the top of the brain slice, but there is limited resolution of tumour cell invasion into the slice. Our novel imaging and quantification technique involves embedding stained brain slices into an agar block, re-sectioning the slice in the Z-direction onto slides, and then using confocal microscopy to image cellular invasion into the brain tissue. This imaging technique allows for the visualization of invasive structures beneath the spheroid that would otherwise go undetected using traditional microscopy approaches. Our ImageJ macro (BraInZ) allows for the quantification of GBM brain slice invasion in the Z-direction. Importantly, we note striking differences in the modes of motility observed when GBM cells invade into Matrigel in vitro versus into brain tissue ex vivo highlighting the importance of incorporating the brain microenvironment when studying GBM invasion. In summary, our version of the ex vivo brain slice invasion assay improves upon previously published models by more clearly differentiating between migration along the top of the brain slice versus invasion into the slice.

Phenotype-based screening can identify small molecules that elicit a desired cellular response, but additional approaches are required to characterize their targets and mechanisms of action. Here, we show that a compound termed LCS3, which selectively impairs the growth of human lung adenocarcinoma (LUAD) cells, induces oxidative stress. To identify the target that mediates this effect, we use thermal proteome profiling (TPP) and uncover the disulfide reductases GSR and TXNRD1 as targets. We confirm through enzymatic assays that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identifies NQO1 loss as a mechanism of LCS3 resistance. This work highlights the ability of TPP to uncover targets of small molecules identified by high-throughput screens and demonstrates the potential therapeutic utility of inhibiting disulfide reductases in LUAD.

Metastatic breast cancer is challenging to effectively treat, highlighting the need for an improved understanding of host factors that influence metastatic tumor cell colonization and growth in distant tissues. The lungs are a common site of breast cancer metastasis and are host to a population of tissue-resident eosinophils. Eosinophils are granulocytic innate immune cells known for their prominent roles in allergy and Th2 immunity. Though their presence in solid tumors and metastases have been reported for decades, the influence of eosinophils on metastatic tumor growth in the lungs is unclear. We used transgenic mouse models characterized by elevated pulmonary eosinophils (IL5Tg mice) and eosinophil-deficiency (ΔdblGATA mice), as well as antibody-mediated depletion of eosinophils, to study the role of eosinophils in EO771 mammary tumor growth in the lungs. We found that IL5Tg mice exhibit reduced pulmonary metastatic colonization and decreased metastatic tumor burden compared to wild-type (WT) mice or eosinophil-deficient mice. Eosinophils co-cultured with tumor cellsex vivoproduced peroxidase activity and induced tumor cell death, indicating that eosinophils are capable of releasing eosinophil peroxidase (EPX) and killing EO771 tumor cells. We found that lung eosinophils expressed phenotypic markers of activation during EO771 tumor growth in the lungs, and that metastatic growth was accelerated in eosinophil-deficient mice and in WT mice after immunological depletion of eosinophils. Our results highlight an important role for eosinophils in restricting mammary tumor cell growth in the lungs and support further work to determine whether strategies to trigger local eosinophil degranulation may decrease pulmonary metastatic growth.

MEK inhibitors (MEKi) have limited efficacy in KRAS mutant lung adenocarcinoma (LUAD) patients, and this is attributed to both intrinsic and adaptive mechanisms of drug resistance. While many studies have focused on the former, there remains a dearth of data regarding acquired resistance to MEKi in LUAD. We established trametinib-resistant KRAS mutant LUAD cells through dose escalation and performed targeted MSK-IMPACT sequencing to identify drivers of MEKi resistance. Comparing resistant cells to their sensitive counterparts revealed alteration of genes associated with trametinib response. We describe a state of "drug addiction" in resistant cases where cells are dependent on continuous culture in trametinib for survival. We show that dependence on ERK2 suppression underlies this phenomenon and that trametinib removal hyperactivates ERK, resulting in ER stress and apoptosis. Amplification of KRASG12C occurs in drug-addicted cells and blocking mutant-specific activity with AMG 510 rescues the lethality associated with trametinib withdrawal. Furthermore, we show that increased KRASG12C expression is lethal to other KRAS mutant LUAD cells, consequential to ERK hyperactivation. Our study determines the drug-addicted phenotype in lung cancer is associated with KRAS amplification and demonstrates that toxic acquired genetic changes can develop de novo in the background of MAPK suppression with MEK inhibitors. We suggest that the presence of mutant KRAS amplification in patients may identify those that may benefit from a "drug holiday" to circumvent drug resistance. These findings demonstrate the toxic potential of hyperactive ERK signaling and highlight potential therapeutic opportunities in patients bearing KRAS mutations.

β-Cells respond to metabolic demand with functional compensation, followed by decompensation and failure, but it is not clear what controls this progression. We examined if anti-apoptotic Bcl-xL prevents β-cell decompensation under chronic glucose excess.

Hypoxia develops in germinal centers (GCs) induced by model antigens; however, it is unknown whether tumor-reactive GCs are also hypoxic. We identified GC hypoxia in lymph nodes (LNs) draining murine mammary tumors and lethally irradiated tumor cells, and found that hypoxia is associated with the levels of antibody-secreting B cells. Hypoxic culture conditions impaired the proliferation of activated B cells, and inhibited class-switching to IgG1 and IgA immunoglobulin isotypes in vitro. To assess the role of the hypoxic response in tumor-reactive GCs in vivo, we deleted von Hippel-Lindau factor (VHL) in class-switched B cells and found decreased GC B cells in tumor-draining LNs, reduced class-switched and tumor-specific antibodies in the circulation, and modified phenotypes of tumor-infiltrating T cells and macrophages. We also detected the hypoxia marker carbonic anhydrase IX in the GCs of LNs from breast cancer patients, providing evidence that GC hypoxia develops in humans. We conclude that GC hypoxia develops in TDLNs, and that the hypoxic response negatively regulates tumor-induced humoral immune responses in preclinical models.

Current immunotherapies for lung cancer are only effective in a subset of patients. Identifying tumor-derived factors that facilitate immunosuppression offers the opportunity to develop novel strategies to supplement and improve current therapeutics. We sought to determine whether expression of driver oncogenes in lung cancer cells affects cytokine secretion, alters the local immune environment, and influences lung tumor progression. We demonstrate that oncogenic EGFR and KRAS mutations, which are early events in lung tumourigenesis, can drive cytokine and chemokine production by cancer cells. One of the most prominent changes was in CCL5, which was rapidly induced by KRASG12V or EGFRL858R expression, through MAPK activation. Immunocompetent mice implanted with syngeneic KRAS-mutant lung cancer cells deficient in CCL5 have decreased regulatory T cells (Tregs), evidence of T cell exhaustion, and reduced lung tumor burden, indicating tumor-cell CCL5 production contributes to an immune suppressive environment in the lungs. Furthermore, high CCL5 expression correlates with poor prognosis, immunosuppressive regulatory T cells, and alteration to CD8 effector function in lung adenocarcinoma patients. Our data support targeting CCL5 or CCL5 receptors on immune suppressive cells to prevent formation of an immune suppressive tumor microenvironment that promotes lung cancer progression and immunotherapy insensitivity.

ABSTRACT In the progression of diabetes, pancreatic islet β-cells respond to increased metabolic demand with functional compensation, followed by pathogenic decompensation of mitochondria-dependent insulin secretion. It is not clear what mechanisms drive, or control, mitochondrial decompensation. Here, we report that anti-apoptotic Bcl-x L maintains mitochondrial integrity in β-cells under non-apoptotic levels of glucose stress. Prolonged glucose excess causes transcriptional reprogramming of glycolysis and β-cell identity genes, while sensitizing glucose-stimulated Ca 2+ signaling and insulin secretion. Deletion of Bcl-x L amplifies this insulin hypersecretion and increases mitochondrial fusion, mitochondrial volume, and oxygen consumption, whereas ATP-coupled respiration and mitochondrial hyperpolarization become impaired. Of note, Bcl-x L -deficient β-cells have impaired Pgc-1α expression, and develop specific defects in the expression of Tfam, mitochondrial ribosomal genes, and OXPHOS components under glucose stress. Bcl-x L limits high glucose-induced mitochondrial ROS (mitoROS) levels and pharmacological normalization of mitoROS in Bcl-x L KO cells rescues glucose-induced defects in mitochondrial gene expression and changes to β-cell identity. Our data identify mitoROS as a primary retrograde driver of transcriptional re-wiring in β-cells exposed to excess glucose, and reveal Bcl-x L as an important safeguard against transcriptional and functional decompensation of β-cell mitochondria. Bcl-x L and mitoROS may thus be viable targets to prevent early β-cell dysfunction and the progression of diabetes.

Lineage transformation between lung cancer subtypes is a poorly understood phenomenon associated with resistance to treatment and poor patient outcomes. Here, we aimed to model this transition to define underlying biological mechanisms and identify potential avenues for therapeutic intervention. Small cell lung cancer (SCLC) is neuroendocrine in identity and, in contrast to non-SCLC (NSCLC), rarely contains mutations that drive the MAPK pathway. Likewise, NSCLCs that transform to SCLC concomitantly with development of therapy resistance downregulate MAPK signaling, suggesting an inverse relationship between pathway activation and lineage state. To test this, we activated MAPK in SCLC through conditional expression of mutant KRAS or EGFR, which revealed suppression of the neuroendocrine differentiation program via ERK. We found that ERK induces the expression of ETS factors that mediate transformation into a NSCLC-like state. ATAC-seq demonstrated ERK-driven changes in chromatin accessibility at putative regulatory regions and global chromatin rewiring at neuroendocrine and ETS transcriptional targets. Further, ERK-mediated induction of ETS factors as well as suppression of neuroendocrine differentiation were dependent on histone acetyltransferase activities of CBP/p300. Overall, we describe how the ERK-CBP/p300-ETS axis promotes a lineage shift between neuroendocrine and non-neuroendocrine lung cancer phenotypes and provide rationale for the disruption of this program during transformation-driven resistance to targeted therapy.

High-throughput phenotype-based screening of large libraries of novel compounds without known targets can identify small molecules that elicit a desired cellular response, but additional approaches are required to find and characterize their targets and mechanisms of action. Here we show that a compound termed lung cancer screen 3 (LCS3), previously selected for its ability to impair the growth of human lung adenocarcinoma (LUAD) cell lines, but not normal lung cells, induces oxidative stress and activates the NRF2 signaling pathway by generating reactive oxygen species (ROS) in sensitive LUAD cell lines. To identify the target that mediates this effect, we applied thermal proteome profiling (TPP) and uncovered the disulfide reductases GSR and TXNRD1 as LCS3 targets. Through enzymatic assays using purified protein, we confirmed that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are correspondingly sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identified the loss of NQO1 as a mechanism of LCS3 resistance. This work highlights the ability of TPP to uncover targets of small molecules identified by high-throughput screens and demonstrates the potential utility of inhibiting disulfide reductases as a therapeutic strategy for LUAD.

Abstract Introduction: Glioblastoma multiforme (GBM) is a grade IV brain tumor that is considered incurable with a median overall survival of 15 months. This low survival rate is partially attributed to the highly infiltrative nature of GBM cells. A key component of GBM cell invasion is the degradation of extracellular matrix (ECM), which is facilitated by the formation of invadosomes: focal adhesions (FAs) and invadopodia. The majority of GBM patients will receive a conventional radiation schedule of 2 Gy in 30 fractions, while a subset of patients will receive a hypofractioned radiation schedule of ~3 Gy in 15 fractions. Recent pre-clinical reports indicate the population of GBM cells that survive ionizing radiation (IR) exhibit increased invasiveness, suggesting IR may regulate FA and invadopodia function. Understanding how IR affects invadosome biology may lead to new therapeutic strategies to halt the invasion of GBM cells and increase overall survival of patients treated with radiation. Results: We found that irradiated GBM cells were significantly more invasive in vitro. GBM cells irradiated with 0-4 Gy and plated onto fluorescently-tagged gelatin to analyze ECM degradation. Cells exposed to 0-2 Gy IR produced small dot-like patterns of degradation resembling active invadopodia. However, cells exposed to 3-4 Gy IR produced oblong-shaped patterns of degradation at the periphery of the cell resembling active FAs. Using immunofluorescent markers of invadopodia (TKS5/actin) and FAs (vinculin), we found total focal adhesion and invadopodia formation remained unchanged. Rather, we found that exposing cells to 3 Gy IR increased pFAK-397 in FA structures suggesting IR increases ECM degradation by promoting FA activity. Conclusions: These data suggest higher IR doses promote FA-mediated ECM degradation and GBM cell invasion. We are currently investigating the impact of IR on FAs, ECM degradation, and GBM cell invasion in murine brain slices ex vivo. Citation Format: Lisa R. Decotret, Rocky Shi, Kevin L. Bennewith. Targeting focal adhesions to limit radiation-induced glioblastoma cell invasion [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-053.