Glioblastoma (GBM) is the most common and aggressive primary central nervous system malignancy. Significant resistance to therapeutic intervention is a core feature of GBM that drives tumor recurrence and underlies the remarkably poor clinical outcomes associated with this disease. This review explores the therapeutic strategies and molecular pathways involved in GBM. Therapy resistance in GBM depends on multiple interconnected macrostructural and biomolecular mechanisms, including aggressive and diffuse invasion, tumor microtube network formation, stem-like cell enrichment, selective neurovascular permeability, an immunosuppressive microenvironment, and a high degree of inter- and intra-tumoral heterogeneity. Collectively, these pathobiological features insulate specific tumor compartments and maintain GBM viability despite significant treatment-induced cellular stress. While there is enthusiasm for addressing GBM therapeutic resistance, the scale of the challenge remains immense. The identified resistance mechanisms extensively interact and can compensate for single-target assaults. Emerging overlaps between neuro-oncology and developmental neurobiology additionally suggest that GBM may exploit therapeutic resistance mechanisms yet to be identified, functioning beyond the current scientific understanding. Thus, the scope and diversity of this problem demand a comprehensive therapeutic approach capable of targeting multiple interacting mechanisms of therapeutic resistance.

Abstract Glioblastoma multiforme (GBM) is the most common adult primary brain malignancy. Aggressive, diffuse invasion is regulated by a dynamic cytoskeleton and significantly contributes to poor survival. Intraparenchymal tumor cell dissemination prevents complete surgical resection and compromises essential brain functions. Invasive cells are inherently less sensitive to traditional cytotoxic therapies. Recent investigation into targeted therapies has failed to address invasion/cytoskeleton based GBM therapy resistance. Furthermore, established GBM cell line modeling in 2 or 3 dimensions (3D) does not capture the genetic diversity or clinical relevance of patient derived primary (1') GBM cell lines, which may uncover/predict differential responses in tumors to novel therapies. Cytoskeleton remodeling and cell invasion in patient-derived primary GBM cells generates unique structures called tumor microtubes (TMs) not seen in established, cultured GBM cell lines. TMs are ultralong, pro-invasive, tubulin-enriched protrusions that promote chemo- and radioresistance in GBM patient derived primary cells. Using our extensive GBM cell line biobank derived from 1' and/or therapy resistant recurrent (2') tumors, we investigated the effects of a novel drug targeting the microtubule cytoskeleton. Plinabulin is a tubulin targeting agent that crosses the blood brain barrier and demonstrated anticancer efficacy in Phase III trial patients with non-small cell lung carcinoma. Plinabulin also has antiproliferative properties in 2D GBM cells. Here, we evaluate the efficacy of plinabulin treatment upon 3D neurosphere viability and invasion in 5 GBM 1' and 2' recurrent patient derived cell lines. Within 24h of matrigel embedding, 3D GBM patient neurospheres generate robust pro-invasive TMs. Plinabulin treatment (10-30 nM) at embedding followed by drug washout after 24h eliminated existing TMs and inhibited single cell invasion (80% relative to controls) in both 1' and 2' GBM patient neurospheres. Cell survival was significantly reduced in plinabulin treated neurospheres (30 nM). Plinabulin efficacy was evaluated in a clinically relevant therapeutic ITR model: GBM patient neurospheres invaded (I) for 48h to establish TM networks; were treated (T) with 1-50 nM plinabulin for 24h prior to drug washout; and recovered (R) for 96h. In 1', 2', or matched 1' and 2' cell line pairs from the same patient, 10-40 nM plinabulin sustained invasion inhibition (>90%) through 96h post washout and completely blocked TM extension relative to controls, consistent with established clinical concentrations of plinabulin. This study reveals plinabulin as a potent GBM therapeutic targeting pro-invasion TM networks and survival in 1' and therapy resistant 2' GBM patient cell lines. Citation Format: Kathryn M. Eisenmann, Kenneth Lloyd, Krista M. Pettee, Kathryn M. Becker, Jason L. Schroeder, Kevin Reinard, James R. Tonra, Ramon Mohanlol, Lan Huang. Plinabulin, a novel tubulin targeting agent, collapses the tumor microtube network in primary and recurrent patient derived glioblastoma cell lines to inhibit neurosphere invasion and survival [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A009.

This study aimed to understand extracellular mechanical stimuli's effect on prostate cancer cells' metastatic progression within a three-dimensional (3D) bone-like microenvironment. In this study, a mechanical loading platform, EQUicycler, has been employed to create physiologically relevant static and cyclic mechanical stimuli to a prostate cancer cell (PC-3)-embedded 3D tissue matrix. Three mechanical stimuli conditions were applied: control (no loading), cyclic (1% strain at 1 Hz), and static mechanical stimuli (1% strain). The changes in prostate cancer cells' cytoskeletal reorganization, polarity (elongation index), proliferation, expression level of N-Cadherin (metastasis-associated gene), and migratory potential within the 3D collagen structures were assessed upon mechanical stimuli. The results have shown that static mechanical stimuli increased the metastasis progression factors, including cell elongation (p < 0.001), cellular F-actin accumulation (p < 0.001), actin polymerization (p < 0.001), N-Cadherin gene expression, and invasion capacity of PC-3 cells within a bone-like microenvironment compared to its cyclic and control loading counterparts. This study established a novel system for studying metastatic cancer cells within bone and enables the creation of biomimetic in vitro models for cancer research and mechanobiology.

The Coronavirus disease 2019 (COVID-19) re-shaped patient care in the United States beginning in March 2020. While fear of contracting the virus was prominent within the general population, hospitals also prioritized surges of COVID-19 patients by canceling in-person clinic appointments, clinical trials, and elective surgeries. To evaluate the state of clinical practice during the first wave of COVID-19, a regional survey was conducted of clinicians from the University of Toledo Medical Center (UTMC) and ProMedica Toledo hospitals and area clinics from March 9 to July 31, 2020. Qualitative free-form responses from clinicians indicated that both hospital systems observed decreases in patient loads and canceled clinics. We then evaluated how COVID-19 impacted workload in specialty clinics specifically within UTMC. Clinical productivity changes were quantified by evaluating Work Relative Value Units (wRVUs) for UTMC clinics. wRVUs compared to the same period in 2019 revealed the pandemic’s effects of suppressing wRVU in nearly all clinics examined in the initial stages of the first wave. wRVUs recovered to 2019 levels in most specialties and even surpassed 2019 levels by the end of the first wave of the pandemic. The recovery of wRVUs within specialty care during the first wave of the COVID-19 pandemic reveals the adaptability of the UTMC medical system in Northwest Ohio for navigating a rapidly changing infectious disease landscape.

Glioblastoma (GBM) is a progressive and lethal brain cancer. Malignant control of actin and microtubule cytoskeletal mechanics facilitates two major GBM therapeutic resistance strategies-diffuse invasion and tumor microtube network formation. Actin and microtubule reorganization is controlled by Rho-GTPases, which exert their effects through downstream effector protein activation, including Rho-associated kinases (ROCK) 1 and 2 and mammalian diaphanous-related (mDia) formins (mDia1, 2, and 3). Precise spatial and temporal balancing of the activity between these effectors dictates cell shape, adhesion turnover, and motility. Using small molecules targeting mDia, we demonstrated that global agonism (IMM02) was superior to antagonism (SMIFH2) as anti-invasion strategies in GBM spheroids. Here, we use IDH-wild-type GBM patient-derived cell models and a novel semi-adherent in vitro system to investigate the relationship between ROCK and mDia in invasion and tumor microtube networks. IMM02-mediated mDia agonism disrupts invasion in GBM patient-derived spheroid models, in part by inducing mDia expression loss and tumor microtube network collapse. Pharmacological disruption of ROCK prevented invasive cell-body movement away from GBM spheres, yet induced ultralong, phenotypically abnormal tumor microtube formation. Simultaneously targeting mDia and ROCK did not enhance the anti-invasive/-tumor microtube effects of IMM02. Our data reveal that targeting mDia is a viable GBM anti-invasion/-tumor microtube networking strategy, while ROCK inhibition is contraindicated.

Invasive motility limits treatment efficacy and is a significant contributor to poor outcomes in glioblastoma (GBM). GBM tumor microtubes are actin- and microtubule-enriched membrane tubes that facilitate invasive motility and underlie many components of GBM pathophysiology. Rho-GTPases mediate GBM invasion through localized activation of cytoskeletal effector proteins, such as mammalian Diaphanous-related formins (mDia) and Rho-associated protein kinase (ROCK). Active mDia nucleates and polymerizes F-actin and independently stabilizes microtubules, while active ROCK phosphorylates myosin phosphatase and myosin light chain to induce the actomyosin crosslinking required for contractility. GBM invasion depends on a delicate balance between mDia-mediated extension of leading-edge structures (such as tumor microtubes) and ROCK-mediated contraction of trailing cell bodies. In this study, we assessed the roles of ROCK and mDia in tumor microtube-associated GBM invasion using a 3D patient-derived neurosphere model of GBM invasion. Neurospheres were embedded in 3D matrices and treated with the small molecule inhibitor of ROCK (Y-27632) and the small molecule agonist of mDia (IMM-02). Treatment with Y-27632 alone reduced the total distance of cell body migration and increased tumor microtube length without effecting the total area of neurosphere invasion. Tumor microtubes extending from Y-27632 treated neurospheres displayed an atypical undulant morphology, further suggesting that ROCK inhibition modifies primary invasion programs. Treatment with IMM-02 alone profoundly reduced total area of neurosphere invasion, distance of cell body migration, and length of tumor microtubes. Combination treatment effects (Y-27632 + IMM-02) were time dependent. At 24 hours, combination treatment did not significantly reduce total area of invasion over either individual treatment. However, tumor microtubes were shorter with combination treatment than Y-27632 alone and longer than IMM-02 alone. At 96 hours, combination treatment reduced total area invaded and length of tumor microtubes in comparison to Y-27632 alone, but no significant difference was observed in comparison to IMM-02 alone. Western blot analysis of both free-floating patient-derived 3D neurospheres and semi-adherent “2.5D” monolayer cultures demonstrated that agonism of mDia formins with IMM-02 (96-hrs) results in the progressive loss of mDia1 and mDia2 protein expression. Therefore, endogenous mDia regulatory mechanisms triggered in response to continuous agonist-mediated mDia activation may inhibit mDia function more effectively than direct antagonism strategies in GBM. Conclusively, both IMM-02 and Y-27632 treatment disrupt the tumor microtube mechanism of GBM neurosphere invasion. Further studies are warranted to evaluate these small molecule compounds for potential anti-tumor microtube therapeutic effect in vivo. Citation Format: Kathryn N. Becker, Krista M. Pettee, Amanda Sugrue, Kevin A. Reinard, Jason L. Schroeder, Kathryn M. Eisenmann. ROCK and mDia have dynamic roles in glioblastoma tumor microtube invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2887.

Background Epithelial ovarian cancer (EOC) cells disseminate within the peritoneal cavity, in part, via the peritoneal fluid as single cells, clusters, or spheroids. Initial single cell egress from a tumor can involve disruption of cell-cell adhesions as cells are shed from the primary tumor into the peritoneum. In epithelial cells, Adherens Junctions (AJs) are characterized by homotypic linkage of E-cadherins on the plasma membranes of adjacent cells. AJs are anchored to the intracellular actin cytoskeletal network through a complex involving E-cadherin, p120 catenin, β-catenin, and αE-catenin. However, the specific players involved in the interaction between the junctional E-cadherin complex and the underlying F-actin network remains unclear. Recent evidence indicates that mammalian Diaphanous-related (mDia) formins plays a key role in epithelial cell AJ formation and maintenance through generation of linear actin filaments. Binding of αE-catenin to linear F-actin inhibits association of the branched-actin nucleator Arp2/3, while favoring linear F-actin bundling. We previously demonstrated that loss of mDia2 was associated with invasive single cell egress from EOC spheroids through disruption of junctional F-actin.Results In the current study, we now show that mDia2 has a role at adherens junctions (AJs) in EOC OVCA429 cells and human embryonic kidney (HEK) 293 cells through its association with αE-catenin and β-catenin. mDia2 depletion in EOC cells leads to reduction in actin polymerization and disruption of cell-cell junctions with decreased interaction between β-catenin and E-cadherin.Conclusions Our results support a necessary role for mDia2 in AJ stability in EOC cell monolayers and indicate a critical role for mDia formins in regulating EOC AJs during invasive transitions.* 3D : three dimensional AJ : adherens junction CytoD : cytochalasin D EMT : epithelial mesenchymal transition EOC : epithelial ovarian cancer FH2 : formin homology 2 HEK : human embryonic kidney IF : immunofluorescence IP : immunoprecipitation KD : knockdown mDia : mammalian Diaphanous PLA : proximity ligation assay SMIFH2 : small molecule inhibitor of FH2

High-grade glioma (HGG, WHO Grade III–IV) accounts for the majority of adult primary malignant brain tumors. Failure of current therapies to target invasive glioma cells partly explains the minimal survival advantages: invasive tumors lack easily-defined surgical margins, and are inherently more chemo- and radioresistant. Much work centers upon Rho GTPase-mediated glioma invasion, yet downstream Rho effector roles are poorly understood and represent potential therapeutic targets. The roles for the mammalian Diaphanous (mDia)-related formin family of Rho effectors have emerged in invasive/metastatic disease. mDias assemble linear F-actin to promote protrusive cytoskeletal structures underlying tumor cell invasion. Small molecule mDia intramimic (IMM) agonists induced mDia functional activities including F-actin polymerization. mDia agonism inhibited polarized migration in Glioblastoma (WHO Grade IV) cells in three-dimensional (3D) in vitro and rat brain slice models. Here, we evaluate whether clinically-relevant high-grade glioma patient-derived neuro-sphere invasion is sensitive to formin agonism. Surgical HGG samples were dissociated, briefly grown as monolayers, and spontaneously formed non-adherent neuro-spheres. IMM treatment dramatically inhibited HGG patient neuro-sphere invasion, both at neuro-sphere embedding and mid-invasion assay, inducing an amoeboid morphology in neuro-sphere edge cells, while inhibiting actin- and tubulin-enriched tumor microtube formation. Thus, mDia agonism effectively disrupts multiple aspects of patient-derived HGG neuro-sphere invasion.

Non-thermal plasma has been a promising new cancer treatment modality in plasma oncology field. It generates extracellular and intracellular reactive species which are key factors for the treatment of cancer cells. In this study, we investigated the differential effect of non-thermal plasma on both A549 lung adenocarcinoma and MRC-5 lung fibroblast cells. Extracellular generation of reactive species in both A549 lung cancer and MRC-5 normal lung fibroblast cells were similar, whereas intracellular penetration of reactive species generated by plasma in A549 cancer cells were almost fourfold higher than the normal cells. Interestingly, A549 cancer cells treated for shorter (15 and 30 s) and longer durations (60 and 120 s) get arrested in S-phase (19%) and G2/M phase (28%) respectively. In a healthy MRC-5 cells, few cells arrested in S and G2/M phase in relative to A549 cells for all treatment time. Finally, we evaluated the expression of apoptosis-related genes, H2AX, BAX, P53, Caspase-8, and ATM on normal and cancer cells. There was a higher expression of BAX gene for 120 s plasma treated A549 cell samples at day 1 relative to MRC-5 cells. These findings demonstrate that non-thermal plasma generated reactive species creates intracellular stress, that arrests cell cycle and induces apoptosis in cancer cells. This study suggests that non-thermal plasma could be a potential therapy for lung cancer treatment.

Due to high rates of recurrent metastasis during EOC progression, there is a critical unmet need to limit advancement of metastatic lesions present at diagnosis and upon initial recurrence. In non-hematogenous dissemination, cancer cells metastasize within the peritoneum and attach to visceral organs. Single cells, multi-cell clusters, or highly invasive multi-cellular spheroids are shed into peritoneal fluid from primary tumors, or are released by mechanical disruption of peritoneal metastases during cytoreductive surgery. Peritoneal EOC metastasis is not halted upon surgical removal of primary or metastatic tumors; it is a dynamic, longitudinal process of recurring tumors in the peritoneal cavity/pleural space. Recurrent peritoneal metastasis likely occurs via tumor cells suspended in the accumulating ascites fluid attaching to and invading the peritoneal mesothelium. Thus, EOC spheroids promote recurrent peritoneal metastasis and must be targeted for therapeutic intervention. We wished to assess the therapeutic utility of small molecules called intramimics (IMM) and Diaphanous-autoregulatory domain (DAD) in EOC spheroid peritoneal metastasis models. Both IMMs and DADs stimulate mDia2 formin function, and are thus classified as agonists. Formins are intracellular cytoskeletal nanomachines that assemble G-actin monomers into F-actin filaments. Formin-mediated F-actin assembly regulates stress fiber formation and generates forces to deform membranes into protrusive structures (i.e., lamellae, filopodia) to enable cell migration. While this process is necessary for normal cell migration (i.e., wound healing, development), it is abnormally invoked in metastasis. mDia proteins also support tumor cell-cell interactions by promoting the assembly of the F-actin architecture underlying cell-cell adherens junctions (AJs) that link cells of epithelial sheets together. IMMs and DADs specifically agonize mDia2 formins, keeping the nanomachine “on”, and in so doing, promote constitutive F-actin assembly within cells. Therefore, we hypothesize that mDia2 formin agonism would effectively block EOC spheroid invasion in 3D matrices by halting single cell invasive egress from spheroids. Using SKOV-3 and OVCA429 adenocarcinoma EOC cells, we demonstrated that mDia2 protein associates with proteins underlying AJ complexes. Specifically, mDia2 protein associates with alpha and beta catenins, yet not E- or N-cadherins. We further show that siRNA-mediated depletion of mDia2 alters the subcellular localization of AJ-associated proteins, impacting F-actin accumulation at the AJ and enhancing spheroid invasive dissemination. In contrast, mDia agonism using IMM and mDia DAD small molecules effectively inhibits EOC spheroid invasion, disallowing single cell egress from spheroids. Collectively, these results indicate a role for mDia formins in regulating invasive egress from EOC spheroids. Thus, there may be therapeutic utility to targeting mDia formin cytoskeleton effectors in halting EOC spheroid invasive egress. Citation Format: Krista M. Pettee, Yuqi Zhang, Kathryn M. Eisenmann. TARGETING THE FORMIN-ASSEMBLED ACTIN CYTOSKELETON AS AN ANTI-INVASION STRATEGY IN OVARIAN CANCER SPHEROIDS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-024.

The tumor microenvironment (TME) promotes tumor cell invasion and metastasis. An important step in the shift to a pro-cancerous microenvironment is the transformation of normal stromal fibroblasts to carcinoma-associated fibroblasts (CAFs). CAFs are present in a majority of solid tumors and can directly promote tumor cell motility via cytokine, chemokine and growth factor secretion into the TME. The exact effects that the TME has upon cytoskeletal regulation in motile tumor cells remain enigmatic. The conserved formin family of cytoskeleton regulating proteins plays an essential role in the assembly and/or bundling of unbranched actin filaments. Mammalian Diaphanous-related formin 2 (mDia2/DIAPH3/Drf3/Dia) assembles a dynamic F-actin cytoskeleton that underlies tumor cell migration and invasion. We therefore sought to understand whether CAF-derived chemokines impact breast tumor cell motility through modification of the formin-assembled F-actin cytoskeleton. In MDA-MB-231 cells, conditioned media (CM) from WS19T CAFs, a human breast tumor-adjacent CAF line, significantly and robustly increased wound closure and invasion relative to normal human mammary fibroblast (HMF)-CM. WS19T-CM also promoted proteasome-mediated mDia2 degradation in MDA-MB-231 cells relative to control HMF-CM and WS21T CAF-CM, a breast CAF cell line that failed to promote robust MDA-MB-231 migration. Cytokine array analysis of CM identified up-regulated secreted factors in WS19T relative to control WS21T CM. We identified CXCL12 as a CM factor influencing loss of mDia2 protein while increasing MDA-MB-231 cell migration. Our data suggest a mechanism whereby CAFs promote tumor cell migration and invasion through CXCL12 secretion to regulate the mDia2-directed cytoskeleton in breast tumor cells.

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults and aggressive treatment only extends survival by months. While metastasis outside the CNS is rare, GBM is highly invasive. Failure of current GBM therapies to target invasive cells partly explains why these treatments confer only minimal survival advantages: invasive tumors lack easily-defined margins, making complete surgical resection impossible, and invasive GBM cells are inherently more chemo- and radioresistant. Therefore, anti-invasive therapies may effectively sensitize GBM cells to conventional therapies and improve survival. Anti-invasive treatments are thus greatly needed, and cellular mechanisms governing GBM invasion represent understudied therapeutic targets. Much work has centered upon how Rho GTPases mediate GBM invasion, yet the roles of downstream Rho effector proteins are poorly understood and represent potential novel therapeutic targets. A role for the mammalian Diaphanous (mDia)-related formin family of Rho GTPase effector proteins has emerged in metastatic disease. mDias are nanomachines generating linear actin filaments to drive protrusive cytoskeletal structures underlying tumor cell invasion. Using novel small molecule mDia agonists (IMMs, orn intramimics) that induce endogenous mDia functional activities, including F-actin polymerization, we demonstrated roles for mDia in driving polarized GBM cell migration. mDia agonism halted GBM spheroid invasion in three-dimensional (3D) in vitro and ex vivo rat brain slice models. Here, we evaluate if GBM patient cell lines are sensitive to formin agonism to halt invasion. Four patient-derived GBM cell lines were isolated as single cell suspensions, and spontaneously formed non-adherent neurospheres. Neurospheres were embedded in 3D-matrices and allowed to invade +/- IMMs. IMMs dramatically inhibited GBM patient neurosphere invasion, significantly impacting both distance single cells migrated from neurosphere edges and lengths of actin-enriched cellular extensions into matrices. Thus, mDia agonism effectively disrupted multiple aspects of patient-derived GBM neurosphere invasion in vitro, warranting further investigation in patient-derived xenografts.

The mammalian Diaphanous-related (mDia) formins are cytoskeletal regulators that assemble and, in some cases, bundle filamentous actin (F-actin), as well as stabilize microtubules. The development of small molecule antagonists and agonists that interrogate mDia formin function has allowed us to investigate the roles of formins in disease states. A small molecule inhibitor of FH2 domain (SMIFH2) inhibits mDia-dependent actin dynamics and abrogates tumor cell migration and cell division in vitro and ex vivo tissue explants. mDia formin activation with small molecule intramimics IMM01/02 and mDia2-DAD peptides inhibited glioblastoma motility and invasion in vitro and ex vivo rat brain slices. However, SMIFH2, IMMs, and mDia2 DAD efficacy in vivo remains largely unexplored and potential toxicity across a range of developmental phenotypes has not been thoroughly characterized. In this study, we performed an in vivo screen of early life-stage toxicity in Danio rerio zebrafish embryos 2 days post-fertilization (dpf) in response to SMIFH2, IMM01/02, and mDia2 DAD. SMIFH2 at concentrations ≥5–10 μM induced significant defects in developing zebrafish, including shorter body lengths, tail curvature and defective tail cellularity, craniofacial malformations, pericardial edema, absent and/or compromised vasculature function and flow, depressed heart rates and increased mortality. Conversely, IMM and mDia2 DAD peptides were minimally toxic at concentrations up to 10–20 and 50 μM, respectively. SMIFH2's therapeutic potential may therefore be limited by its substantial in vivo toxicity at functional concentrations. mDia formin agonism with IMMs and mDia2 DADs may therefore be a more effective and less toxic anti-invasive therapeutic approach.

The inhibition of apoptosis, disruption of cellular microtubule dynamics, and over-activation of the epithelial mesenchymal transition (EMT), are involved in the progression, metastasis, and resistance of colorectal cancer (CRC) to chemotherapy. Therefore, the design of a molecule that can target these pathways could be an effective strategy to reverse CRC progression and metastasis. In this study, twelve novel silybin derivatives, HM015a-HM015k (15a-15k) and compound 17, were screened for cytotoxicity in CRC cell lines. Compounds HM015j and HM015k (15k and 15j) significantly decreased cell proliferation, inhibited colony formation, and produced cell cycle arrest in CRC cells. Furthermore, 15k significantly induced the formation of reactive oxygen species and apoptosis. It induced the cleavage of the intrinsic apoptotic protein (Bax p21) to its more efficacious fragment, p18. Compound 15k also inhibited tubulin expression and disrupted its structure. Compound 15k significantly decreased metastatic LOVO cell migration and invasion. Furthermore, 15k reversed mesenchymal morphology in HCT116 and LOVO cells. Additionally, 15k significantly inhibited the expression of the mesenchymal marker N-cadherin and upregulated the expression of the epithelial marker, E-cadherin. Compound 15k inhibited the expression of key proteins known to induce EMT (i.e., DVL3, β-catenin, c-Myc) and upregulated the anti-metastatic protein, cyclin B1. Overall, in vitro, 15k significantly inhibited CRC progression and metastasis by inhibiting apoptosis, tubulin activity and the EMT pathways. Overall, these data suggest that compound 15k should be tested in vivo in a CRC animal model for further development.

Recent breakthroughs in plasma medicine have identified a potential application for the non-thermal plasma in cancer therapy. Most studies on the effects of non-thermal plasma on cancer cells have used traditional two-dimensional (2D) monolayer cell culture. However, very few studies are conducted employing non-thermal plasma in animal models. Two dimensional models do not fully mimic the three-dimensional (3D) tumor microenvironment and animal models are expensive and time-consuming. Therefore, we used 3D collagen matrices that closely resemble the native geometry of cancer tissues and provide more physiologically relevant results than 2D models, while providing a more cost effective and efficient precursor to animal studies. We previously demonstrated a role for non-thermal plasma application in promoting apoptotic cell death and reducing the viability of A549 lung adenocarcinoma epithelial cells cultured upon 2D matrices. In this study, we wished to determine the efficacy of non-thermal plasma application in driving apoptotic cell death of A549 lung cancer cells encapsulated within a 3D collagen matrix. The percentage of apoptosis increased as treatment time increased and was time dependent. In addition, the anti-viability effect of plasma was demonstrated. Twenty-four hours post-plasma treatment, 38% and 99% of cell death occurred with shortest (15 s) and longest treatment time (120 s) respectively at the plasmatreated region. We found that plasma has a greater effect on the viability of A549 lung cancer cells on the superficial surface of 3D matrices and has diminishing effects as it penetrates the 3D matrix. We also identified the nitrogen and oxygen species generated by plasma and characterized their penetration in vertical and lateral directions within the 3D matrix from the center of the plasma-treated region. Therefore, the utility of non-thermal dielectric barrier discharge plasma in driving apoptosis and reducing the viability of lung cancer cells in 3D collagen matrix indicates a therapeutic potential that warrants further research.

Morphological plasticity in response to environmental cues in migrating cancer cells requires F-actin cytoskeletal rearrangements. Conserved formin family proteins play critical roles in cell shape, tumor cell motility, invasion and metastasis, in part, through assembly of non-branched actin filaments. Diaphanous-related formin-2 (mDia2/Diaph3/Drf3/Dia) regulates mesenchymal-to-amoeboid morphological conversions and non-apoptotic blebbing in tumor cells by interacting with its inhibitor diaphanous-interacting protein (DIP), and disrupting cortical F-actin assembly and bundling. F-actin disruption is initiated by a CXCL12-dependent mechanism. Downstream CXCL12 signaling partners inducing mDia2-dependent amoeboid conversions remain enigmatic. We found in MDA-MB-231 tumor cells CXCL12 induces DIP and mDia2 interaction in blebs, and engages its receptor CXCR4 to induce RhoA-dependent blebbing. mDia2 and CXCR4 associate in blebs upon CXCL12 stimulation. Both CXCR4 and RhoA are required for CXCL12-induced blebbing. Neither CXCR7 nor other Rho GTPases that activate mDia2 are required for CXCL12-induced blebbing. The Rho Guanine Nucleotide Exchange Factor (GEF) Net1 is required for CXCL12-driven RhoA activation and subsequent blebbing. These results reveal CXCL12 signaling, through CXCR4, directs a Net1/RhoA/mDia-dependent signaling hub to drive cytoskeleton rearrangements to regulate morphological plasticity in tumor cells. These signaling hubs may be conserved during normal and cancer cells responding to chemotactic cues.

Traditional cancer treatments like radiotherapy and chemotherapy have drawbacks and are not selective for killing only cancer cells. Nonthermal atmospheric pressure plasmas with dielectric barrier discharge (DBD) can be applied to living cells and tissues and have emerged as novel tools for localized cancer therapy. The purpose of this study was to investigate the different effects caused by miniature DBD (mDBD) plasma to A549 lung cancer cells. In this study, A549 lung cancer cells cultured in 12 well plates were treated with mDBD plasma for specified treatment times to assess the changes in the size of the area of cell detachment, the viability of attached or detached cells, and cell migration. Furthermore, we investigated an innovative mDBD plasma-based therapy for localized treatment of lung cancer cells through apoptotic induction. Our results indicate that plasma treatment for 120 sec causes apoptotic cell death in 35.8% of cells, while mDBD plasma treatment for 60 sec, 30 sec, or 15 sec causes apoptotic cell death in 20.5%, 14.1%, and 6.3% of the cell population, respectively. Additionally, we observed reduced A549 cell migration in response to mDBD plasma treatment. Thus, mDBD plasma system can be a viable platform for localized lung cancer therapy.

Abstract The tumor microenvironment (TME) is a heterogeneous region comprised of tumor cells, stromal cells, and secreted factors that make the environment favorable for cancer formation and progression. An important step in the shift to a pro-cancerous environment is the transformation of normal stromal fibroblasts to carcinoma-associated fibroblasts (CAFs). CAFs are a critical cell type to understand. They are present in a majority of solid tumors and can have a direct effect to enhance adjacent tumor cell motility via their innate ability to secret cytokines, chemokine and growth factors into the TME. We sought to understand how CAF-derived chemokines impact breast tumor cell motility through modification of the F-actin cytoskeleton. We collected cultured media (CM) from WS19T fibroblasts, a patient-derived breast tumor CAF cell line. CAF-CM dramatically enhanced wound-closure in MDA-MB-231 monolayers, relative to normal fibroblast cultured media. The chemokine CXCL12 (SDF1 alpha) has been identified as a potential secreted CAF-directed signal driving tumor cell migration. To determine if CXCL12 was the soluble factor in CAF-CM promoting tumor cell migration, MDA-MB-231 cells were preincubated with AMD3100, an inhibitor of the CXCL12 receptor, CXCR4 prior to CAF-CM application and wounding. AMD3100 effectively blocked CAF-CM-induced MDA-MB-231 migration. CXCL12 incubation with MDA-MB-231 cells equally promoted MDA-MB-231 wound closure, supporting the notion that CXCL12 is secreted by CAFs to promote MDA-MB-231 motility. We previously showed a link between CXCL12 directed CXCR4 signaling and a critical regulator of the dynamic F-actin cytoskeleton, mammalian Diaphanous-related formin (mDia2). This formin regulates the assembly and bundling of unbranched actin filaments and plays a role in tumor cell migration and invasion programs. Western blotting CAF-CM-treated MDA-MB-231 cells revealed a near complete loss of mDia2 protein. mDia1 and ROCK expression were unaffected with CAF-CM treatment. These data were consistent with treatment with a functional mDia FH2-domain inhibitor SMIFH2, which likewise suppresses mDia2 protein levels in MDA-MB-231 cells. It remains unclear if CAF-derived CXCL12 first suppresses mDia2 FH2 activity, leading to loss of mDia2 protein, thus promoting tumor cell motility. Current experiments are designed to address this gap in the knowledge, potentially indicating a role for CAF-derived soluble factors in modulating mDia2 protein function and expression in migrating tumor cells. Citation Format: Kaitlyn Dvorak, Kathryn M. Eisenmann. Carcinoma-associated fibroblast-derived CXCL12 enhances MDA-MB-231 tumor cell migration though mDia2 formin suppression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5068.