Browsing by Author "Elzey, Bennett D."
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Item A novel, safe, fast and efficient treatment for Her2-positive and negative bladder cancer utilizing an EGF-anthrax toxin chimera(Wiley, 2020) Jack, Sherwin; Madhivanan, Kayalvizhi; Ramadesika, Swetha; Subramanian, Sneha; Edwards, Daniel F., II; Elzey, Bennett D.; Dhawan, Deepika; McCluskey, Andrew; Kischuk, Erin M.; Loftis, Alexander R.; Truex, Nicholas; Santos, Michael; Lu, Mike; Rabideau, Amy; Pentelute, Bradley; Collier, John; Kaimakliotis, Hristos; Koch, Michael; Ratliff, Timothy L.; Knapp, Deborah W.; Aguilar, Ruben C.; Urology, School of MedicineBladder cancer is the sixth most common cancer in the United States, and it exhibits an alarming 70% recurrence rate. Thus, the development of more efficient antibladder cancer approaches is a high priority. Accordingly, this work provides the basis for a transformative anticancer strategy that takes advantage of the unique characteristics of the bladder. Unlike mucin-shielded normal bladder cells, cancer cells are exposed to the bladder lumen and overexpress EGFR. Therefore, we used an EGF-conjugated anthrax toxin that after targeting EGFR was internalized and triggered apoptosis in exposed bladder cancer cells. This unique agent presented advantages over other EGF-based technologies and other toxin-derivatives. In contrast to known agents, this EGF-toxin conjugate promoted its own uptake via receptor microclustering even in the presence of Her2 and induced cell death with a LC50 < 1 nM. Furthermore, our data showed that exposures as short as ≈3 min were enough to commit human (T24), mouse (MB49) and canine (primary) bladder cancer cells to apoptosis. Exposure of tumor-free mice and dogs with the agent resulted in no toxicity. In addition, the EGF-toxin was able to eliminate cells from human patient tumor samples. Importantly, the administration of EGF-toxin to dogs with spontaneous bladder cancer, who had failed or were not eligible for other therapies, resulted in ~30% average tumor reduction after one treatment cycle. Because of its in vitro and in vivo high efficiency, fast action (reducing treatment time from hours to minutes) and safety, we propose that this EGF–anthrax toxin conjugate provides the basis for new, transformative approaches against bladder cancer.Item Bone Microenvironment-Suppressed T Cells Increase Osteoclast Formation and Osteolytic Bone Metastases in Mice(Wiley, 2022) Arellano, Danna L.; Juárez, Patricia; Verdugo-Meza, Andrea; Almeida-Luna, Paloma S.; Corral-Avila, Juan A.; Drescher, Florian; Olvera, Felipe; Jiménez, Samanta; Elzey, Bennett D.; Guise, Theresa A.; Fournier, Pierrick G.J.; Medicine, School of MedicineImmunotherapies use components of the immune system, such as T cells, to fight cancer cells, and are changing cancer treatment, causing durable responses in some patients. Bone metastases are a debilitating complication in advanced breast and prostate cancer patients. Approved treatments fail to cure bone metastases or increase patient survival and it remains unclear whether immunotherapy could benefit patients. The bone microenvironment combines various immunosuppressive factors, and combined with T cell products could increase bone resorption fueling the vicious cycle of bone metastases. Using syngeneic mouse models, our study revealed that bone metastases from 4T1 breast cancer contain tumor-infiltrating lymphocyte (TILs) and their development is increased in normal mice compared to immunodeficient and T-cell depleted mice. This effect seemed caused by the TILs specifically in bone, because T-cell depletion increased 4T1 orthotopic tumors and did not affect bone metastases from RM-1 prostate cancer cells, which lack TILs. T cells increased osteoclast formation ex vivo and in vivo contributing to bone metastasis vicious cycle. This pro-osteoclastic effect is specific to unactivated T cells, because activated T cells, secreting interferon γ (IFNγ) and interleukin 4 (IL-4), actually suppressed osteoclastogenesis, which could benefit patients. However, non-activated T cells from bone metastases could not be activated in ex vivo cultures. 4T1 bone metastases were associated with an increase of functional polymorphonuclear and monocytic myeloid-derived suppressor cells (MDSCs), potent T-cell suppressors. Although effective in other models, sildenafil and zoledronic acid did not affect MDSCs in bone metastases. Seeking other therapeutic targets, we found that monocytic MDSCs are more potent suppressors than polymorphonuclear MDSCs, expressing programmed cell death receptor-1 ligand (PD-L1)+ in bone, which could trigger T-cell suppression because 70% express its receptor, programmed cell death receptor-1 (PD-1). Collectively, our findings identified a new mechanism by which suppressed T cells increase osteoclastogenesis and bone metastases. Our results also provide a rationale for using immunotherapy because T-cell activation would increase their anti-cancer and their anti-osteoclastic properties.Item Chemically-defined generation of human hemogenic endothelium and definitive hematopoietic progenitor cells(Elsevier, 2022) Chang, Yun; Syahirah, Ramizah; Oprescu, Stephanie N.; Wang, Xuepeng; Jung, Juhyung; Cooper, Scott H.; Torregrosa-Allen, Sandra; Elzey, Bennett D.; Hsu, Alan Y.; Randolph, Lauren N.; Sun, Yufei; Kuang, Shihuan; Broxmeyer, Hal E.; Deng, Qing; Lian, Xiaojun; Bao, Xiaoping; Microbiology and Immunology, School of MedicineHuman hematopoietic stem cells (HSCs), which arise from aorta-gonad-mesonephros (AGM), are widely used to treat blood diseases and cancers. However, a technique for their robust generation in vitro is still missing. Here we show temporal manipulation of Wnt signaling is sufficient and essential to induce AGM-like hematopoiesis from human pluripotent stem cells. TGFβ inhibition at the stage of aorta-like SOX17+CD235a- hemogenic endothelium yielded AGM-like hematopoietic progenitors, which closely resembled primary cord blood HSCs at the transcriptional level and contained diverse lineage-primed progenitor populations via single cell RNA-sequencing analysis. Notably, the resulting definitive cells presented lymphoid and myeloid potential in vitro; and could home to a definitive hematopoietic site in zebrafish and rescue bloodless zebrafish after transplantation. Engraftment and multilineage repopulating activities were also observed in mouse recipients. Together, our work provided a chemically-defined and feeder-free culture platform for scalable generation of AGM-like hematopoietic progenitor cells, leading to enhanced production of functional blood and immune cells for various therapeutic applications.Item Effect of Paclitaxel Stereochemistry on X-Ray-Triggered Release of Paclitaxel from CaWO4/Paclitaxel-Coloaded PEG-PLA Nanoparticles(ACS, 2022) Sarkar, Kaustabh; Torregrossa-Allen, Sandra E.; Elzey, Bennett D.; Narayanan, Sanjeev; Langer, Mark P.; Durm, Gregory A.; Won, You-Yeon; Radiation Oncology, School of MedicineFor many locally advanced tumors, the chemotherapy-radiotherapy (CT-RT) combination ("chemoradiation") is currently the standard of care. Intratumoral (IT) CT-based chemoradiation has the potential to overcome the limitations of conventional systemic CT-RT (side effects). For maximizing the benefits of IT CT-RT, our laboratory has previously developed a radiation-controlled drug release formulation, in which anticancer drug paclitaxel (PTX) and radioluminescent CaWO4 (CWO) nanoparticles (NPs) are co-encapsulated with poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) block copolymers ("PEG-PLA/CWO/PTX NPs"). These PEG-PLA/CWO/PTX NPs enable radiation-controlled release of PTX and are capable of producing sustained therapeutic effects lasting for at least one month following a single IT injection. The present article focuses on discussing our recent finding about the effect of the stereochemical structure of PTX on the efficacy of this PEG-PLA/CWO/PTX NP formulation. Stereochemical differences in two different PTX compounds ("PTX-S" from Samyang Biopharmaceuticals and "PTX-B" from Biotang) were characterized by 2D heteronuclear/homonuclear NMR, Raman spectroscopy, and circular dichroism measurements. The difference in PTX stereochemistry was found to significantly influence their water solubility (WS); PTX-S (WS ≈ 4.69 μg/mL) is about 19 times more water soluble than PTX-B (WS ≈ 0.25 μg/mL). The two PTX compounds showed similar cancer cell-killing performances in vitro when used as free drugs. However, the subtle stereochemical difference significantly influenced their X-ray-triggered release kinetics from the PEG-PLA/CWO/PTX NPs; the more water-soluble PTX-S was released faster than the less water-soluble PTX-B. This difference was manifested in the IT pharmacokinetics and eventually in the survival percentages of test animals (mice) treated with PEG-PLA/CWO/PTX NPs + X-rays in an in vivo human tumor xenograft study; at short times (<1 month), concurrent PEG-PLA/CWO/PTX-S NPs produced a greater tumor-suppression effect, whereas PEG-PLA/CWO/PTX-B NPs had a longer-lasting radio-sensitizing effect. This study demonstrates the importance of the stereochemistry of a drug in a therapy based on a controlled release formulation.Item Engineered natural killer cells impede the immunometabolic CD73-adenosine axis in solid tumors(eLife Sciences, 2022-07-11) Chambers, Andrea M.; Lupo, Kyle B.; Wang, Jiao; Cao, Jingming; Utturkar, Sagar; Lanman, Nadia; Bernal-Crespo, Victor; Jalal, Shadia; Pine, Sharon R.; Torregrosa-Allen, Sandra; Elzey, Bennett D.; Matosevic, Sandro; Medicine, School of MedicineImmunometabolic reprogramming due to adenosine produced by CD73 (encoded by the 5'-ectonucleotidase gene NT5E) is a recognized immunosuppressive mechanism contributing to immune evasion in solid tumors. Adenosine is not only known to contribute to tumor progression, but it has specific roles in driving dysfunction of immune cells, including natural killer (NK) cells. Here, we engineered human NK cells to directly target the CD73-adenosine axis by blocking the enzymatic activity of CD73. In doing so, the engineered NK cells not only impaired adenosinergic metabolism driven by the hypoxic uptake of ATP by cancer cells in a model of non-small-cell lung cancer, but also mediated killing of tumor cells due to the specific recognition of overexpressed CD73. This resulted in a 'single agent' immunotherapy that combines antibody specificity, blockade of purinergic signaling, and killing of targets mediated by NK cells. We also showed that CD73-targeted NK cells are potent in vivo and result in tumor arrest, while promoting NK cell infiltration into CD73+ tumors and enhancing intratumoral activation.Item Engineering chimeric antigen receptor neutrophils from human pluripotent stem cells for targeted cancer immunotherapy(Cell Press, 2022) Chang, Yun; Syahirah, Ramizah; Wang, Xuepeng; Jin, Gyuhyung; Torregrosa-Allen, Sandra; Elzey, Bennett D.; Hummel, Sydney N.; Wang, Tianqi; Li, Can; Lian, Xiaojun; Deng, Qing; Broxmeyer, Hal E.; Bao, Xiaoping; Microbiology and Immunology, School of MedicineNeutrophils, the most abundant white blood cells in circulation, are closely related to cancer development and progression. Healthy primary neutrophils present potent cytotoxicity against various cancer cell lines through direct contact and via generation of reactive oxygen species. However, due to their short half-life and resistance to genetic modification, neutrophils have not yet been engineered with chimeric antigen receptors (CARs) to enhance their antitumor cytotoxicity for targeted immunotherapy. Here, we genetically engineered human pluripotent stem cells with synthetic CARs and differentiated them into functional neutrophils by implementing a chemically defined platform. The resulting CAR neutrophils present superior and specific cytotoxicity against tumor cells both in vitro and in vivo. Collectively, we established a robust platform for massive production of CAR neutrophils, paving the way to myeloid cell-based therapeutic strategies that would boost current cancer-treatment approaches.Item Epigenetic aberrations of gene expression in a rat model of hepatocellular carcinoma(Taylor & Francis, 2022) Boycott, Cayla; Beetch, Megan; Yang, Tony; Lubecka, Katarzyna; Ma, Yuexi; Zhang, Jiaxi; Kurzava Kendall, Lucinda; Ullmer, Melissa; Ramsey, Benjamin S.; Torregrosa-Allen, Sandra; Elzey, Bennett D.; Cox, Abigail; Lanman, Nadia Atallah; Hui, Alisa; Villanueva, Nathaniel; de Conti, Aline; Huan, Tao; Pogribny, Igor; Stefanska, Barbara; Anatomy, Cell Biology and Physiology, School of MedicineHepatocellular carcinoma (HCC) is mostly triggered by environmental and life-style factors and may involve epigenetic aberrations. However, a comprehensive documentation of the link between the dysregulated epigenome, transcriptome, and liver carcinogenesis is lacking. In the present study, Fischer-344 rats were fed a choline-deficient (CDAA, cancer group) or choline-sufficient (CSAA, healthy group) L-amino acid-defined diet. At the end of 52 weeks, transcriptomic alterations in livers of rats with HCC tumours and healthy livers were investigated by RNA sequencing. DNA methylation and gene expression were assessed by pyrosequencing and quantitative reverse-transcription PCR (qRT-PCR), respectively. We discovered 1,848 genes that were significantly differentially expressed in livers of rats with HCC tumours (CDAA) as compared with healthy livers (CSAA). Upregulated genes in the CDAA group were associated with cancer-related functions, whereas macronutrient metabolic processes were enriched by downregulated genes. Changes of highest magnitude were detected in numerous upregulated genes that govern key oncogenic signalling pathways, including Notch, Wnt, Hedgehog, and extracellular matrix degradation. We further detected perturbations in DNA methylating and demethylating enzymes, which was reflected in decreased global DNA methylation and increased global DNA hydroxymethylation. Four selected upregulated candidates, Mmp12, Jag1, Wnt4, and Smo, demonstrated promoter hypomethylation with the most profound decrease in Mmp12. MMP12 was also strongly overexpressed and hypomethylated in human HCC HepG2 cells as compared with primary hepatocytes, which coincided with binding of Ten-eleven translocation 1 (TET1). Our findings provide comprehensive evidence for gene expression changes and dysregulated epigenome in HCC pathogenesis, potentially revealing novel targets for HCC prevention/treatment.Item Folate Receptor Beta Designates Immunosuppressive Tumor-Associated Myeloid Cells That Can Be Reprogrammed with Folate-Targeted Drugs(AACR, 2021-02) Cresswell, Gregory M.; Wang, Bingbing; Kischuk, Erin M.; Broman, Meaghan M.; Alfar, Rami A.; Vickman, Renee E.; Dimitrov, Dimiter S.; Kularatne, Sumith A.; Sundaram, Chandru P.; Singhal, Sunil; Eruslanov, Evgeniy B.; Crist, Scott A.; Elzey, Bennett D.; Ratliff, Timothy L.; Low, Philip S.; Urology, School of MedicineAlthough immunotherapies of tumors have demonstrated promise for altering the progression of malignancies, immunotherapies have been limited by an immunosuppressive tumor microenvironment (TME) that prevents infiltrating immune cells from performing their anticancer functions. Prominent among immunosuppressive cells are myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) that inhibit T cells via release of immunosuppressive cytokines and engagement of checkpoint receptors. Here, we explore the properties of MDSCs and TAMs from freshly isolated mouse and human tumors and find that an immunosuppressive subset of these cells can be distinguished from the nonimmunosuppressive population by its upregulation of folate receptor beta (FRβ) within the TME and its restriction to the TME. This FRβ+ subpopulation could be selectively targeted with folate-linked drugs. Delivery of a folate-targeted TLR7 agonist to these cells (i) reduced their immunosuppressive function, (ii) increased CD8+ T-cell infiltration, (iii) enhanced M1/M2 macrophage ratios, (iv) inhibited tumor growth, (v) blocked tumor metastasis, and (vi) improved overall survival without demonstrable toxicity. These data reveal a broadly applicable strategy across tumor types for reprogramming MDSCs and TAMs into antitumorigenic immune cells using a drug that would otherwise be too toxic to administer systemically. The data also establish FRβ as the first marker that distinguishes immunosuppressive from nonimmunosuppressive subsets of MDSCs and TAMs. Because all solid tumors accumulate MDSCs and TAMs, a general strategy to both identify and reprogram these cells should be broadly applied in the characterization and treatment of multiple tumors.Item Multispecific targeting of glioblastoma with tumor microenvironment-responsive multifunctional engineered NK cells(National Academy of Science, 2021) Wang, Jiao; Toregrosa-Allen, Sandra; Elzey, Bennett D.; Utturkar, Sagar; Lanman, Nadia Atallah; Bernal-Crespo, Victor; Behymer, Matthew M.; Knipp, Gregory T.; Yun, Yeonhee; Veronesi, Michael C.; Sinn, Anthony L.; Pollok, Karen E.; Brutkiewicz, Randy R.; Nevel, Kathryn S.; Matosevic, Sandro; Radiology and Imaging Sciences, School of MedicineTumor antigen heterogeneity, a severely immunosuppressive tumor microenvironment (TME) and lymphopenia resulting in inadequate immune intratumoral trafficking, have rendered glioblastoma (GBM) highly resistant to therapy. To address these obstacles, here we describe a unique, sophisticated combinatorial platform for GBM: a cooperative multifunctional immunotherapy based on genetically engineered human natural killer (NK) cells bearing multiple antitumor functions including local tumor responsiveness that addresses key drivers of GBM resistance to therapy: antigen escape, immunometabolic reprogramming of immune responses, and poor immune cell homing. We engineered dual-specific chimeric antigen receptor (CAR) NK cells to bear a third functional moiety that is activated in the GBM TME and addresses immunometabolic suppression of NK cell function: a tumor-specific, locally released antibody fragment which can inhibit the activity of CD73 independently of CAR signaling and decrease the local concentration of adenosine. The multifunctional human NK cells targeted patient-derived GBM xenografts, demonstrated local tumor site-specific activity in the tissue, and potently suppressed adenosine production. We also unveil a complex reorganization of the immunological profile of GBM induced by inhibiting autophagy. Pharmacologic impairment of the autophagic process not only sensitized GBM to antigenic targeting by NK cells but promoted a chemotactic profile favorable to NK infiltration. Taken together, our study demonstrates a promising NK cell-based combinatorial strategy that can target multiple clinically recognized mechanisms of GBM progression simultaneously.Item Piceatannol, a Dietary Polyphenol, Alleviates Adipose Tissue Loss in Pre-Clinical Model of Cancer-Associated Cachexia via Lipolysis Inhibition(MDPI, 2022-05-31) Kershaw, Jonathan C.; Elzey, Bennett D.; Guo, Xiao-Xuan; Kim, Kee-Hong; Urology, School of MedicineCancer-associated cachexia (CAC) is the nutrition-independent loss of lean muscle and adipose tissues, and results in reduced chemotherapy effectiveness and increased mortality. Preventing adipose loss is considered a key target in the early stages of cachexia. Lipolysis is considered the central driver of adipose loss in CAC. We recently found that piceatannol, but not its analogue resveratrol, exhibits an inhibitory effect on lipolysis. The objective of this study was to investigate the role of piceatannol in cancer-associated lipolysis and cachexia-induced weight loss. Cancer cell-induced lipolysis in adipocytes was stimulated using cancer-conditioned media (CCM) or co-culture with human pancreatic cancer cells and the cachexia-associated cytokines TNF-α and interleukin-6 in 3T3-L1 adipocytes. C26 colon carcinoma-bearing mice were modeled using CAC in vivo. Piceatannol reduced cancer-associated lipolysis by at least 50% in both CCM and cytokine-induced lipolysis in vitro. Further gene and protein analysis confirmed that piceatannol modulated the stability of lipolytic proteins. Moreover, piceatannol protected tumor-bearing mice against weight-loss in early stages of CAC largely through preserving adipose tissue, with no effect on survival. This study demonstrates the use of a dietary compound to preserve adipose in models of early stage CAC and provides groundwork for further investigation of piceatannol or piceatannol-rich foods as alternative medicine in the preservation of body fat mass and future CAC therapy.