Life Sciences Commons^™

Modification Of The Tumor Microenvironment Enhances Anti-Pd-1 Immunotherapy In Metastatic Melanoma, Guilan Shi, Megan Scott, Cathryn G. Mangiamele, Richard Heller

Bioelectrics Publications

Resistance to checkpoint-blockade treatments is a challenge in the clinic. Both primary and acquired resistance have become major obstacles, greatly limiting the long-lasting effects and wide application of blockade therapy. Many patients with metastatic melanoma eventually require further therapy. The absence of T-cell infiltration to the tumor site is a well-accepted contributor limiting immune checkpoint inhibitor efficacy. In this study, we combined intratumoral injection of plasmid IL-12 with electrotransfer and anti-PD-1 in metastatic B16F10 melanoma tumor model to increase tumor-infiltrating lymphocytes and improve therapeutic efficacy. We showed that effective anti-tumor responses required a subset of tumor-infiltrating CD8⁺ and CD4 …

Targeting Delivery Of Bcl-2 Family Protein Inhibitor Has The Potential To Treat Cancer And Fibrosis, Mohammad Nurul Huda

Open Access Theses & Dissertations

Apoptosis is a naturally occurring cell death mechanism to remove the selective cell population. B-cell leukemia/lymphoma-2 (BCL-2) family protein plays a critical role in activating the upstream apoptosis signaling pathway, primarily the intrinsic apoptosis pathway. The BCL-2 family consists of both pro-and anti-apoptotic proteins, which are structurally and functionally similar, containing up to four BCL-2 homologies (BH) motifs (BH1-4). Defecting apoptosis along this signaling pathway can lead to various events, including malignant cell transformation, tumor metastasis, tissue fibrosis, and drug resistance. In fibrosis, the aberrant apoptosis signaling also activates multiple effector proteins and growth factors, such as TGF-β, CTGF, and …

Gene Expression Profiling Of Mapk Pathway Inhibitor Resistance In Cutaneous Melanoma: Can Bioinformatics Be Used To Select Better Melanoma Cell Lines?, Stephen Luebker

Theses & Dissertations

Melanoma is the deadliest form of skin cancer, and incidence has continued to increase. Half of all melanomas have a BRAF V600E mutation and respond to MAPK pathway inhibitors, including BRAF inhibitor therapy or BRAF/MEK inhibitor combination therapy, but nearly all patients develop treatment resistance. Melanoma cell lines produce variable results as models of MAPK pathway inhibitor resistance. To better understand how the genomic similarity of a melanoma cell line to patient-derived tumors affects resistance mechanisms, differences in DNA mutations and copy-number alterations were compared between melanoma cell lines profiled by the Cancer Cell Line Encyclopedia and cutaneous melanoma tumors …

Impact Of Intratumor Heterogeneity And The Tumor Microenvironment In Shaping Tumor Evolution And Response To Therapy, Akash Mitra

Dissertations & Theses (Open Access)

Intratumor heterogeneity (ITH) is a crucial challenge in cancer treatment. The genotypic and phenotypic heterogeneity underlying diverse cancer types leads to subclonal variation, which may result in mixed or failed response to therapy. The heterogeneity at the tumor level, along with the tumor microenvironment (TME), often shapes tumor evolution and ultimately clinical outcome. Given that modern treatment paradigms increasingly expose patients with metastatic disease to multiple treatment modalities through the course of their disease, there exists a need to characterize robust and predictive biomarkers of response to therapy. In order to accurately characterize tumor evolution, we need to account for …

Pd-L1 Expression On Circulating Tumor Cells May Be Predictive Of Response To Pembrolizumab In Advanced Melanoma: Results From A Pilot Study, Muhammad K. Khattak, Anna L. Reid, James Freeman, Michelle Pereira, Ashleigh Mcevoy, Johnny Lo, Markus Frank, Tarek Meniawy, Ali Didan, Isaac Spencer, Benhur Amanuel, Michael Millward, Mel Ziman, Elin Gray

Research outputs 2014 to 2021

BACKGROUND: PD-1 inhibitors are routinely used for the treatment of advanced melanoma. This study sought to determine whether PD-L1 expression on circulating tumor cells (CTCs) can serve as a predictive biomarker of clinical benefit and response to treatment with the PD-1 inhibitor pembrolizumab.

METHODS: Blood samples were collected from patients with metastatic melanoma receiving pembrolizumab, prior to treatment and 6-12 weeks after initiation of therapy. Multiparametric flow cytometry was used to identify CTCs and evaluate the expression of PD-L1.

RESULTS: CTCs were detected in 25 of 40 patients (63%). Patients with detectable PD-L1

CONCLUSION: Our results reveal the potential of …

Inter-Tumor Heterogeneity-Melanomas Respond Differently To Gm-Csf-Mediated Activation., Adi Moshe, Sivan Izraely, Orit Sagi-Assif, Sapir Malka, Shlomit Ben-Menachem, Tsipi Meshel, Metsada Pasmanik-Chor, Dave Hoon, Isaac P Witz

Articles, Abstracts, and Reports

Granulocyte-monocyte colony stimulating factor (GM-CSF) is used as an adjuvant in various clinical and preclinical studies with contradictory results. These were attributed to opposing effects of GM-CSF on the immune or myeloid systems of the treated patients or to lack of optimal dosing regimens. The results of the present study point to inter-tumor heterogeneity as a possible mechanism accounting for the contrasting responses to GM-CSF incorporating therapies. Employing xenograft models of human melanomas in nude mice developed in our lab, we detected differential functional responses of melanomas from different patients to GM-CSF both in vitro as well as in vivo. …

Il-12 Gene Electrotransfer Triggers A Change In Immune Response Within Mouse Tumors, Guilan Shi, Chelsea Edelblute, Sezgi Arpag, Cathryn Lundberg, Richard Heller

Bioelectrics Publications

Metastatic melanoma is an aggressive skin cancer with a relatively low survival rate. Immune-based therapies have shown promise in the treatment of melanoma, but overall complete response rates are still low. Previous studies have demonstrated the potential of plasmid IL-12 (pIL-12) delivered by gene electrotransfer (GET) to be an effective immunotherapy for melanoma. However, events occurring in the tumor microenvironment following delivery have not been delineated. Therefore, utilizing a B16F10 mouse melanoma model, we evaluated changes in the tumor microenvironment following delivery of pIL-12 using different GET parameters or injection of plasmid alone. The results revealed a unique immune cell …

Abl Kinase Regulation By Braf/Erk And Cooperation With Akt In Melanoma, Aditi Jain, Rakshamani Tripathi, Courtney P. Turpin, Chi Wang, Rina Plattner

Pharmacology and Nutritional Sciences Faculty Publications

The melanoma incidence continues to increase, and the disease remains incurable for many due to its metastatic nature and high rate of therapeutic resistance. In particular, melanomas harboring BRAF^V600E and PTEN mutations often are resistant to current therapies, including BRAF inhibitors (BRAFi) and immune checkpoint inhibitors. Abl kinases (Abl/Arg) are activated in melanomas and drive progression; however, their mechanism of activation has not been established. Here we elucidate a novel link between BRAF^V600E/ERK signaling and Abl kinases. We demonstrate that BRAF^V600E/ERK play a critical role in binding, phosphorylating and regulating Abl localization and Abl/Arg activation …

Paracrine Regulation Of Melanocyte Genomic Stability: A Focus On Nucleotide Excision Repair, Stuart Gordon Jarrett, Katharine Marie Carter, John August D'Orazio

Markey Cancer Center Faculty Publications

UV radiation is a major environmental risk factor for the development of melanoma by causing DNA damage and mutations. Resistance to UV damage is largely determined by the capacity of melanocytes to respond to UV injury by repairing mutagenic photolesions. The nucleotide excision repair (NER) pathway is the major mechanism by which cells correct UV photodamage. This multistep process involves the basic steps of damage recognition, isolation, localized strand unwinding, assembly of a repair complex, excision of the damage‐containing strand 3′ and 5′ to the photolesion, synthesis of a sequence‐appropriate replacement strand, and finally ligation to restore continuity of genomic …

Systematic Analysis Of Whole Exome Sequencing Determines Ret G691s Polymorphism As Germline Variant In Melanoma, Brent J. Smith Jr, Jennifer D. Hintzsche, Carol M. Amato, Aik-Choon Tan, Keith R. Wells, Allison J. Applegate, Rita T. Gonzalez, Jodie R. Barr, William A. Robinson

Marshall Journal of Medicine

Abstract

The RET proto-oncogene encodes a receptor tyrosine kinase that is activated by glial cell derived neutrotrophic factor (GDNF). Previous studies have found that a single nucleotide polymorphism (SNP), RETp (G691S), in the juxtamembrane domain enhances the signaling pathway and promotes tumor growth by GDNF in pancreatic and thyroid cancer in addition to melanoma. It is uncertain however whether this SNP is a germline variant or somatic mutation. A prior study reported that the RETp variant was a germline SNP in desmoplastic and non-desmoplastic melanomas. In the present study, we examined both melanoma tissue samples and matching peripheral blood DNA …

The Soy-Derived Peptide Lunasin Inhibits Invasive Potential Of Melanoma Initiating Cells, Chris Shidal, Jun-Ichi Inaba, Kavitha Yaddanapudi, Keith R. Davis

Plant Pathology Faculty Publications

Lunasin is a 44 amino acid peptide with multiple functional domains including an aspartic acid tail, an RGD domain, and a chromatin-binding helical domain. We recently showed that Lunasin induced a phenotype switch of cancer initiating cells (CIC) out of the stem compartment by inducing melanocyte-associated differentiation markers while simultaneously reducing stem-cell-associated transcription factors. In the present study, we advance the hypothesis that Lunasin can reduce pools of melanoma cells with stem cell-like properties, and demonstrate that Lunasin treatment effectively inhibits the invasive potential of CICs in vitro as well as in vivo in a mouse experimental metastasis model. Mice …

Alternative Methods For The Treatment Of Chemo-Resistant Cancers, Kaitlyn Wong

Doctoral Dissertations

Great strides have been made in cancer therapy in the past century, yet it remains one of the leading causes of death in the United States today. This work aimed to shed light on novel methods to treat a variety of aggressive and often chemo-resistant cancers both in vitro and in vivo. The first aim of this work was to evaluate the therapeutic efficacy of poly(methacryloyloxyethyl phosphorylcholine) (polyMPC) prodrugs compared to standard chemotherapeutic agents. Conjugation of polyMPC to drugs such as doxorubicin (Dox) can result in its improved solubility, prolonged half-life and therapeutic efficacy. PolyMPC and polyMPC-Dox (at a …

Autoantibodies To The Ny-Eso-1 Tumor Antigen In Metastatic Melanoma: Sialylation Of The Fc Region Of Immunoglobulin G Induces Differential Expression Signatures Of Inflammatory Molecules During Dendritic Cell Differentiation And Maturation, Martin Oaks, Nathaniel Rein, John O. Richards, James Shaffer

Journal of Patient-Centered Research and Reviews

Purpose: We tested the hypothesis that different glycoforms of antibodies from patients with metastatic melanoma have different functional effects on human dendritic cell differentiation and maturation.

Methods: Antibodies to the cancer antigen NY-ESO-1 were affinity-purified from patients with melanoma and further fractionated into different glycoforms by lectin chromatography. Sialic acid-rich and sialic acid-poor fractions of these immunoglobulin G antibodies (IgG) were added to dendritic cell cultures during both differentiation and maturation, and the resulting cellular messenger RNA (mRNA) and culture supernatants were tested by microarray and enzyme-linked immunoassay for molecules related to inflammatory pathways.

Results: We identified unique mRNA and …

Electrotransfer Of Single-Stranded Or Double-Stranded Dna Induces Complete Regression Of Palpable B16.F10 Mouse Melanomas, Loree Heller, Vesba Todorovic, Maja Cemazar

Bioelectrics Publications

Enhanced tumor delivery of plasmid DNA with electric pulses in vivo has been confirmed in many preclinical models. Intratumor electrotransfer of plasmids encoding therapeutic molecules has reached Phase II clinical trials. In multiple preclinical studies, a reduction in tumor growth, increased survival or complete tumor regression have been observed in control groups in which vector or backbone plasmid DNA electrotransfer was performed. This study explores factors that could produce this antitumor effect. The specific electrotransfer pulse protocol employed significantly potentiated the regression. Tumor regression was observed after delivery of single-stranded or double-stranded DNA with or without CpG motifs in both …

Characterization Of Differentiation And Prognostic Biomarkers On Cd8+ Tumor-Infiltrating Lymphocytes In Metastatic Melanoma, Richard C. Wu

Dissertations & Theses (Open Access)

CD8⁺ cytotoxic T lymphocytes (CTL) frequently infiltrate tumors, yet most melanoma patients fail to undergo tumor regression. We studied the differentiation of the CD8⁺ tumor-infiltrating lymphocytes (TIL) from 44 metastatic melanoma patients using known T-cell differentiation markers. We also compared CD8⁺ TIL against the T cells from matched melanoma patients’ peripheral blood. We discovered a novel subset of CD8⁺ TIL co-expressing early-differentiation markers, CD27, CD28, and a late/senescent CTL differentiation marker, CD57. This CD8⁺CD57⁺ TIL expressed a cytolytic enzyme, granzyme B (GB), yet did not express another cytolytic pore-forming molecule, perforin (Perf). In …

Histone Deacetylases As Targets For Melanoma Immunotherapy, David Michael Woods

USF Tampa Graduate Theses and Dissertations

Cancer represents the second leading cause of death in the United States. For many malignancies, currently available treatment options offer little long-lasting survival benefits to patients. However, recent studies have shown immunotherapeutic approaches to be an attractive strategy to cancer treatment. While many current immunotherapeutic strategies convey durable responses, such responses are only seen in a minority of patients. An increased understanding of the mechanisms governing tumor immunogenicity and the biology of immune responses is crucial to improving upon the efficacy of current and future cancer immunotherapies. Histone deacetylases (HDACs), enzymes classically associated with regulation of gene expression, have been …

Metastatic Disease: Interactions Between Tumor Cells And Host Environment During Cancer Cell Spread, Jennifer M. Maclean

Electronic Thesis and Dissertation Repository

Tumor and metastasis formation are not cell autonomous phenomena, but rather an evolution of disease within and responding to the host environment. Metastatic spread from a primary tumor occurs as a result of a complex interplay between tumor cells and the host, wherein tumor cells must escape the primary tumor, enter the host vasculature, travel to and arrest in a distant tissue and survive and grow in that new organ. It is known that cells that progress through these stages must both escape and exploit host systems, yet the mechanisms used are not fully understood. Therefore, the goal of this …

Plasmid Injection And Application Of Electric Pulses Alter Endogenous Mrna And Protein Expression In B16.F10 Mouse Melanomas, L. C. Heller, Y. L. Cruz, B. Ferraro, H. Yang, R. Heller

Bioelectrics Publications

The application of electric pulses to tissues causes cell membrane destabilization, allowing exogenous molecules to enter the cells. This delivery technique can be used for plasmid gene therapy. Reporter gene expression after plasmid delivery with eight representative published protocols was compared in B16.F10 mouse melanoma tumors. This expression varied significantly based on the pulse parameters utilized for delivery. To observe the possible influence of plasmid injection and/or pulse application on endogenous gene expression, levels of stress-related mRNAs 4 and 24 h after delivery were determined by PCR array. Increases in mRNA levels for several inflammatory chemokines and cytokines were observed …

Pdest Fg12-Cmv Dsred Vector, Jarod Wolffis, Sheri L. Holmen

Undergraduate Research Opportunities Program (UROP)

Melanoma is the most rapidly increasing malignancy among young people in the United States. If detected early, the disease is easily treated; however, once the disease has metastasized it is largely refractory to conventional therapies and is associated with a high mortality rate. The development of human cancer from a pre-malignant primary tumor to a metastatic lesion that develops at secondary sites is thought to be a multi-step process, requiring many genetic and epigenetic events that provide a growth advantage to cells. It is still unclear which of the many genetic changes in human cancers are required for metastasis. Therefore, …

Nanosecond Pulsed Electric Fields Induce A Mitochondria-Independent Apoptosis In B16f10 Melanoma Cells In Vitro, Wentia Elissa Ford

Theses and Dissertations in Biomedical Sciences

Nanosecond pulsed electric fields (nsPEFs) are ultra-short pulses that induce direct electric field and biological effects that initiate apoptosis. Here the application of ten 300ns pulses ranging in electric fields from 12kV/cm-60kV/cm was administered to determine the effects on B16F10 melanoma cells evaluated by in vitro studies. Initial application of nsPEFs demonstrated apoptosis induction in an electric field- and pulse number-dependent manner measured by caspase activation that correlated with decrease in cell viability 24hr post pulse. In addition caspase activity was shown to be independent of calcium mobilization though ions may play a part in other aspects of apoptosis. The …

In Vivo Murine Melanoma Tumor Responses To Nanosecond Pulsed Electric Field Treatment, Xinhua Chen

Theses and Dissertations in Biomedical Sciences

High intensity nanosecond pulsed electric fields (nsPEF) were applied to melanoma tumors to observe functional and structural biological changes and to investigate the possible molecular mechanisms responsible. An animal model was set up by injecting B16F10 mouse melanoma cells into SKH-1 mice. A treatment (Tx) of 100 pulses: 300 nanosecond duration; 40 kV/cm field strength; at 0.5 Hz rate were delivered to melanoma tumors in 120 mice. The nsPEF Txcaused tumor self-destruction with sharply decreased cell volumes and shrunken nuclei. The apoptotic biochemical tests confirmed nsPEF Tx induced apoptosis in a time-dependent manner. Examination of gross vessel and micro-vessel density …

Evaluation Of Toxicity Following Electrically Mediated Interleukin-12 Gene Delivery In A B16 Mouse Melanoma Model, Loree Heller, Kathleen Merkler, Jeffrey Westover, Yolmari Cruz, Domenico Coppola, Kaaron Benson, Adil Daud, Richard Heller

Bioelectrics Publications

PURPOSE: Interleukin-12 (IL-12) has potential as an immunotherapeutic agent for the treatment of cancer but is unfortunately associated with toxicity. Delivery of a plasmid encoding IL-12 with electroporation induces an antitumor effect in the B16 mouse melanoma model without serious side effects. To translate this observation to the clinic, an evaluation of toxicity was done in the mouse model.

EXPERIMENTAL DESIGN: Weight change, tumor response, blood chemistry and hematology values, and serum IL-12 levels were evaluated. Multiple tissues were analyzed histopathologically.

RESULTS: A pronounced reduction in tumor volume, including a large percentage of complete regressions, was observed after electrically mediated …

Effect Of Electrically Mediated Intratumor And Intramuscular Delivery Of A Plasmid Encoding Ifn Α On Visible B16 Mouse Melanomas, Loree C. Heller, Stephanie F. Ingram, M. Lee Lucas, Richard A. Gilbert, Richard Heller

Bioelectrics Publications

Interferon α may be used as a single agent therapy for metastatic malignant melanoma or as an adjuvant to chemotherapy. Delivery of interferon α by gene therapy offers an alternative to recombinant protein therapy. Electrically mediated delivery enhances plasmid expression in a number of tissues, for instance skin, liver, muscle and tumors including melanomas. Here we compare the effect of delivery of a plasmid encoding mouse interferon α on growth of visible B16 mouse melanomas following electrically mediated delivery to muscle or directly to the tumor. Intratumoral delivery of interferon α plasmid not only slows melanoma growth, but induces complete, …

Il-12 Plasmid Delivery By In Vivo Electroporation For The Successful Treatment Of Established Subcutaneous B16.F10 Melanoma, M. Lee Lucus, Loree Heller, Domenico Coppola, Richard Heller

Bioelectrics Publications

Interleukin-12 (IL-12) has been used in numerous immunotherapy protocols against melanoma. However, delivery of IL-12 in the form of recombinant protein can result in severe toxicity, and gene therapy has had limited success against B16.F10 murine melanoma. The purpose of this study was to examine the effectiveness of in vivo electroporation for the delivery of plasmid DNA encoding IL-12 as an antitumor agent against B16.F10 melanoma. We treated mice bearing established B16.F10 melanoma tumors with intratumoral (i.t.) or intramuscular (i.m.) injections of a plasmid encoding IL-12, followed by in vivo electroporation. For i.t. treatments, we used an applicator containing six …