nci microbial based cancer therapy conference poster session · triple-negative breast cancer...
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NCI Microbial Based Cancer Therapy Conference
POSTER SESSION
Conference sponsored by the National Cancer Institute
Technologies to Overcome Cancer Challenges
July 11-12, 2017 Natcher Center, NIH campus- Building 45, Bethesda, Maryland
POSTER SESSION
No Name Title
Cancer virotherapy
1 Eric Bartee Tumor localized inhibition of the PD1/PDL1 enhances the efficacy of oncolytic myxoma
2 David A. Ornelles MAP3K7 and CHD1 are novel mediators of resistance to VSV oncolysis in prostate cancer
3 Lauren Oldfield Genome-wide modification of herpesvirus genomes using synthetic genomics methods
4 Bernardo A. Mainou Reoviruses Have Enhanced Oncolytic Properties Against Triple-Negative Breast Cancer
5 Liang Deng Intratumoral delivery of inactivated modified vaccinia virus Ankara (iMVA)
6 Liang Deng Intratumoral Delivery of Modified Vaccinia Virus Ankara Expressing Human Flt3L
7 Kate Chiappinelli Epigenetic control of endogenous retroviruses in cancer: implications for immune therapy
8 Biswajit Biswas Therapeutic and Prophylactic Applications of Bacteriophages in Cancer Therapy
9 Ann B. Hill Cytomegalovirus-based vaccines in breast and melanoma mouse tumor models
Bacterial and eukaryotes based cancer therapy
10 Abel Baerga-Ortiz Direct detection of genotoxic or pro-inflammatory bacterial genes in stool samples
11 Adam Fisher Modulation of the Tumor Microenvironment using Synthetic BioticsTM
12 Hassan Brim Gut microbiome analysis reveals dysbiosis in sickle cell diseases patients with veillonella
13 Weiguo Cui Reenergized Adoptive Cell Transfer -A Multi-Pronged Strategy to Treat Solid Tumors
14 David J. Bzik Uncorking the remarkable anti-cancer biology of Toxoplasma
15 Jill Zeilstra-Ryalls Developing a photosynthetic bacterial vector for intratumorial photodynamic therapy
16 Greg Phillips Use of a gnotobiotic mouse model to characterize bacteria/colorectal tumor interactions
17 Wei Kong TRAIL-armed Self-destructing Salmonella serve as “Time-Bombs” to Combat Cancer
18 Bin Xue Multiple Responding Mechanisms in Intestinal Cancer Tissues Mediated by Microbiome
19 Shiladitya DasSarma An Archaeal Therapeutic Drug and Antigen Delivery Employing Proteinaceous Nanoparticles
20 Qiuhong He Loss of Cancer Immune Privilege in Bacterial-based Therapy
21 Roger A Laine Bacterial Secreted Polysaccharide Toxins Bind to Sialin on Tumor Capillary Endothelium
22 Gilad Bachrach Tumor targeting by Fusobacterium nucleatum
23 Mark Gomelsky Optogenetic and chemogenetic platforms for listeria-mediated intratumoral drug delivery
24 Alejandro Alice Vγ9Vδ2 T cells dominate the response to Listeria monocytogenes-based vaccines
25 Shifeng Wang Regulated invasion, chemotaxis, attenuation and endotoxicity of Salmonella
26 Katherine Broadway Contribution of S. enterica Chemotaxis on Mouse Mammary Carcinoma Progression
27 Bahareh Behkam Nanoscale Bacteria-Enabled Autonomous Drug Delivery Systems for Cancer Therapy
28 Yasser Heakal Autophagy and Apoptosis in Triple-Negative Breast Cancer Cells by Heat-labile Enterotoxin
29 Dudley H. McNitt The Streptococcal Adhesin, Scl1, Recognizes Oncofetal Fibronectin
30 Melissa Garcia Dietary fatty acids modulate fungal-host interactions
31 Arturo Ferreira Could Trypanosoma cruzi Infection Be a Good Thing in the Presence of a Tumor?
32 Jesus Vera Combined Parasite-Derived Peptides for Melanoma Therapy
Technologies to Support Research on Microbial based cancer therapy
33 Tsang, Hsiny Cloud-based platform for analyzing TCGA and microbe sequencing data
34 Wenyun Lu Mass spectrometry based metabolomics platform for cancer research
35 Yanming Li Weak Signal Detection of Lung Cancer Risk
0
Cancer virotherapy
1
Tumor localized inhibition of the PD1/PDL1 checkpoint enhances the efficacy of
oncolytic myxoma virus against both local and metastatic melanoma.
Mee Y Bartee, Katherine M Dunlap, Eric Bartee
Medical University of South Carolina, Charleston
While traditional oncolytic therapy is effective at rapidly debulking directly injected tumor masses,
achieving complete eradication of either established local tumors or disseminated metastatic disease
has proven difficult. One potential method to overcome this is to use oncolytic infections to induce
secondary anti-tumor immunotherapy. Unfortunately, while the initial induction of this
immunotherapy is typically robust, its subsequent clinical efficacy is often inhibited through a variety
of immuno-regulatory mechanisms, such as activation of the PD1/PDL1 T-cell checkpoint. In order
to improve the clinical potential of oncolytic therapy, we therefore examined whether inhibition of
the PD1/PDL1 pathway would improve the efficacy of oncolytic myxoma virus against well-
established B16/F10 melanomas.
Our results indicate that systemic inhibition of the PD1/PDL1 pathway during myxoma virus
treatment significantly enhances the efficacy of viral therapy and allows for the complete eradiation
of established melanomas. This increased efficacy was not due to changes in the direct oncolytic
capacity of myxoma virus and instead correlated with enhanced activation of anti-tumor CD8+ T cell
responses. To advance this finding, we further generated a novel oncolytic myxoma virus (vMYX
PD1) which secretes a soluble form of PD1 from infected cells within the tumor microenvironment.
Once secreted, this soluble PD1 binds to PDL1 on the surface of neighboring tumor cells thus
preventing activation of the inhibitory PD1/PDL1 checkpoint. Analysis of this virus indicated that
vMYX-PD1 both induced and maintained CD8+ anti-tumor T-cell responses and that monotherapy
with this virus actually outperformed therapy using the combination of unmodified myxoma and
systemic PD1-blockade. Additionally, localized vMYX-PD1 treatment induced a systemic abscopal
effect capable of significantly reducing established metastatic tumor burden in non-injected
secondary solid organs such as the lung. These results demonstrate that tumor localized inhibition of
the PD1 T-cell checkpoint significantly improves outcomes during oncolytic virotherapy and
establishes a feasible path to translate these findings against clinically relevant disease.
2
MAP3K7 and CHD1 are novel mediators of resistance to VSV oncolysis in prostate cancer
ROBERT S. BAYNE1, DAVID A. ORNELLES2 and DOUGLAS S. LYLES3
1Department of Biochemistry, 2Department of Microbiology and Immunology, Wake Forest School
of Medicine, Winston-Salem, NC
Innate immunity is a prominent determinant of viral pathogenicity and viral tropism; viruses
preferentially infect tissues with abrogated immune responses, such as cancers that downregulated
their antiviral responses. However, some cancers constitutively express antiviral genes and display
viral resistance. This phenomenon will significantly impact the efficacy of several dozen oncolytic
viral therapies currently in clinical trials. We tested the effects of two genes, MAP3K7 and CHD1, on
antiviral gene expression in virus-resistant prostate cancer. These two genes are frequently co-deleted
in high-grade prostate cancers. MAP3K7 and/or CHD1 expression was silenced with shRNA in virus-
resistant PC3 human prostate cancer cells. Transcriptome analysis by RNA-Seq showed that silencing
CHD1 alone and silencing both MAP3K7 and CHD1 decreased antiviral gene expression in PC3 cells,
whereas silencing MAP3K7 alone increased antiviral gene expression. Silencing expression of
MAP3K7, CHD1, or both genes increased susceptibility to infection with oncolytic vesicular
stomatitis virus (VSV). These results indicate that CHD1 plays an important role in the constitutive
expression of antiviral genes. However, the increase in antiviral gene expression in MAP3K7-silencd
cells indicated that the increased viral susceptibility is mediated by other mechanisms. These findings
demonstrate that MAP3K7 and CHD1 regulate viral resistance in prostate cancer. Antiviral gene
expression may be a determinant of viral resistance in prostate cancer, but other mechanisms are
involved as well.
3
Rapid, genome-wide modification of herpesvirus genomes using synthetic genomics methods
Lauren M. Oldfield, Peter Grzesik, Alexander Voorhies, Sanjana Prasad, Nina Alperovich, Derek
MacMath, Claudia Najera, Alexandra Rocha, Diya Chandra, Vladimir Noskov, Michael Montague,
Prashant Desai, Sanjay Vashee
J. Craig Venter Institute
Herpesviruses are clinically important human pathogens and some members of this family, Epstein-
Barr virus (EBV) and Kaposi’s sarcoma-associated virus (KSHV), are oncogenic. The genomes of
herpesviruses range in size between 125 – 250 kb, contain regions of repeated sequence, and have a
high GC content. Historically, manipulation and sequencing of herpesvirus genomes has been
difficult. The cloning of herpesvirus genomes as bacterial artificial chromosomes has greatly
facilitated the manipulation of these DNA sequences using Escherichia coli recombineering,
however, the long timeframe to generate complex mutants and the instability of some herpesvirus
genomes over repeated rounds of mutagenesis still represent significant drawbacks. To improve our
capacity to engineer herpesvirus genomes, we utilized synthetic genomics methods in yeast to clone
overlapping fragments of the herpes simplex virus type 1 (HSV-1) genome and then to assemble the
fragments to regenerate a full-length genome. This modular DNA assembly method allows
researchers to make changes quickly in the genomic fragments in parallel and assemble genomes
using a mixture of wild-type and mutant fragments, rapidly generating mutant HSV-1 genomes with
multiple mutations. Using a number of different techniques, including in vitro CRISPR-Cas9 editing,
we demonstrated the utility of this method by making modifications to a single gene, two genes at a
time and, finally, combinatorial deletions of five tegument genes. HSV-1 was chosen as a model
because infectious virus can be quickly reconstituted in tissue culture and, because of our success
with HSV-1, we have initiated similar genome assemblies of EBV and KSHV. Rapid manipulation
of HSV-1 could also help to engineer effective delivery platforms for therapeutics, such as oncolytic
viruses, or vaccines.
4
Engineered Reoviruses Have Enhanced Oncolytic Properties Against Triple-Negative Breast
Cancer
Roxana M. Rodriguez Stewart1, Angela K. Berger2,3, Jaime Guberman4, and Bernardo A.
Mainou1,2,3
Microbiology and Molecular Genetics1, Department of Pediatrics2, Children’s Healthcare of
Atlanta3, Emory University4, Atlanta, GA 30322
Triple-negative breast cancer (TNBC) constitutes approximately 15% of all breast cancer, has a
higher rate of relapse, and shorter overall survival after metastasis than other subtypes of breast
cancer. There is a need for targeted therapeutics to treat this type of breast cancer, as current therapies
are largely limited to cytotoxic chemotherapy. Mammalian orthoreovirus (reovirus), a nonenveloped
segmented dsRNA virus in the Reoviridae family causes a mostly asymptomatic infection in humans.
Reovirus has been shown to preferentially kill transformed cells and is currently in Phase I-III clinical
trials to assess its efficacy as an oncolytic against a variety of cancers. To engineer reovirus with
enhanced infective and cytopathic properties against triple-negative breast cancer cells, we coinfected
a TNBC cell line (MDA-MB-231) with prototype reoviruses T1L, T2J, and T3D. Following serial
passage, we isolated two reassortant reoviruses, r1Reovirus and r2Reovirus. r1Reovirus and
r2Reovirus contain gene segments predominately from T1L, with one (r2Reovirus) or three
(r1Reovirus) gene segments from T3D and synonymous and nonsynonymous point mutations.
Infection of two TNBC cell lines, MDA-MB-231 and HCC1937, showed that r1Reovirus and
r2Reovirus infect both cell lines more efficiently than T1L and have enhanced capacity to induce cell
death in MDA-MB-231 cells. Although r2Reovirus infected HCC1937 cells more efficiently, all
reovirus serotypes tested impaired cell growth equally without promoting cell death. These data
suggest that r1Reovirus and r2Reovirus encode genomic changes that enhance their ability to infect
TNBC cells. The different inhibitory effects on cell growth by virus infection also suggests reovirus
can impair cancer cell growth through different mechanisms depending on the genetic composition
of the TNBC cells.
5
Intratumoral Delivery of Modified Vaccinia Virus Ankara Expressing Human Flt3L as Cancer
Immunotherapy
Peihong Dai1,2#, Weiyi Wang1#, Ning Yang1#, Stewart Shuman2, Taha Merghoub3,4,5, Jedd D.
Wolchok3,4,5,6, and Liang Deng1,6*
1 Dermatology Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065., 2Molecular
Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065., 3Parker Institute for Cancer
Immunotherapy, Memorial Sloan Kettering Cancer Center, New York, NY 10065., 4Ludwig Center for Cancer Immunotherapy,
Memorial Sloan Kettering Cancer Center, New York, NY 10065., 5Melanoma and Immunotherapeutics Service, Department
of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065, 6Weill Cornell Medical and Graduate Colleges,
New York, NY 10065
Modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia strain that is an important vaccine
vector for infectious diseases and cancers. MVA has a 31-kb deletion of the parental vaccinia genome and
was shown to be safe for human use during smallpox vaccination. The investigation of MVA as cancer
therapeutics has so far been limited to its use as a vaccine vector to express tumor antigens. We
hypothesize that intratumoral delivery of recombinant MVA∆E3L (with deletion of vaccinia virulence
factor E3) expressing human flt3L (Fms-like tyrosine kinase 3 ligand) would provide "in situ therapeutic
vaccine effects." Flt3L plays a critical role in the development of DC subsets, including CD103+/CD8α+
DCs, which are critical for cross-presentation of tumor antigens. We found that MVA∆E3L infection of
B16-F10 and MC38 induces higher levels of IFN-ȕ, IL-6, CCL4 and CCL5 than MVA. MVA∆E3L
induction of type I IFN in cDCs is mainly dependent on the cGAS/STING pathway. Intratumoral injection
of MVA∆E3L is more efficacious than MVA in tumor eradication and extension of survival in bilateral
tumor implantation models, which correlates with stronger induction of activated CD8+ and CD4+ effector
T cells in both injected and non-injected tumors from MVA∆E3L-treated mice compared with MVA-
treated mice. Furthermore, intratumoral injection of MVA∆E3L-hFlt3L exerts stronger anti-tumor effects
than MVA∆E3L in a murine melanoma bilateral implantation model. Our results show that intratumoral
injection of MVA or MVA∆E3L leads to alteration of tumor immune suppressive microenvironment,
which facilitates tumor antigen presentation, recruitment and activation of anti-tumor CD8+ and CD4+ T
cells. Using immune activating virus to produce hFlt3L within tumor microenvironment further enhances
antitumor immune responses. Lastly intratumoral injection of MVA∆E3L-hFlt3L overcomes resistance to
immune checkpoint blockade therapy in murine tumor models. Taken together, our results indicate that
MVA∆E3L-hFlt3L is a promising cancer immunotherapeutic agent.
6
Intratumoral delivery of inactivated modified vaccinia virus Ankara (iMVA) induces systemic
antitumor immunity via STING and Batf3-dependent dendritic cells
Peihong Dai1,2#, Weiyi Wang1#, Ning Yang1, Cristian Serna-Tamayo1, Jacob M. Ricca3, Dmitriy
Zamarin3,4,5,6, Stewart Shuman2, Taha Merghoub3,4,5, Jedd D. Wolchok3,4,5,6, and Liang Deng1,6*
1 Dermatology Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New
York, NY 10065. 2Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York,
NY 10065. 3Parker Institute for Cancer Immunotherapy, Memorial Sloan Kettering Cancer Center,
New York, NY 10065. 4Ludwig Center for Cancer Immunotherapy, Memorial Sloan Kettering
Cancer Center, New York, NY 10065. 5Melanoma and Immunotherapeutics Service, Department of
Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065 6Weill Cornell Medical
and Graduate Colleges, New York, NY 10065
Advanced cancers remain a therapeutic challenge despite recent progress in targeted therapy and
immunotherapy. Novel approaches are needed to alter the tumor immune-suppressive
microenvironment and to facilitate the recognition of tumor antigens that leads to antitumor
immunity. Poxviruses, such as modified vaccinia virus Ankara (MVA), have potential as
immunotherapeutic agents. Here we show that infection of conventional dendritic cells (DCs) with
heat-inactivated or UV-inactivated MVA leads to higher levels of IFN induction than MVA via the
cGAS–STING cytosolic DNA-sensing pathway. Intratumoral injection of inactivated MVA (iMVA)
was effective and generated adaptive antitumor immunity in murine melanoma and colon cancer
models. iMVA-induced antitumor therapy was less effective in STING- or Batf3-deficient mice than
in wild-type mice, indicating that both cytosolic DNA-sensing and Batf3-dependent CD103+/CD8+
DCs are essential for iMVA immunotherapy. The combination of intratumoral delivery of iMVA and
systemic delivery of immune checkpoint blockade generated synergistic antitumor effects in bilateral
tumor implantation models as well as in a unilateral large established tumor model. Our results
suggest that inactivated vaccinia virus could be used as a safe and effective cancer immunotherapeutic
agent for human cancers.
7
Epigenetic control of endogenous retroviruses in cancer: implications for immune therapy
Katherine B. Chiappinelli, PhD, George Washington University Cancer Center
Therapies that activate the host immune system have shown tremendous promise for a wide variety
of solid tumors, with patients exhibiting vigorous and durable responses. However, in most cancer
types, fewer than half of patients respond to these immune therapies. We propose epigenetic therapy
as a mechanism to sensitize these patients. DNA methyltransferase inhibitors (DNMTis) upregulate
immune attraction, including the interferon response, in solid tumors. We have shown that in human
epithelial ovarian cancer cells, DNMTis upregulate viral defense by cytosolic sensing of double-
stranded RNA (dsRNA), triggering a Type I Interferon response and apoptosis. Demethylation and
expression of bidirectionally transcribed endogenous retroviruses (ERVs) is a major component of
the dsRNA that activates the response. Our work shows that treatment with the DNMTi 5-azacytidine
(Aza) sensitizes mouse melanoma cells to subsequent anti-CTLA4 therapy, likely through activation
of the interferon response and subsequent signaling to host immune cells. Our current work aims to
verify this hypothesis. In addition, we observe that adding histone deacetylase inhibitors (HDACis)
to DNMTis can augment the upregulation of specific ERVs and the resulting downstream interferon
response in human cancer cell lines. Specifically, the ERV-K family as well as the Fc2 and ERV-9
families are increased by DNMTi treatment but further augmented by HDACi treatment, while
HDACis alone have minimal effects on the ERVs and the downstream interferon response. We tested
the hypothesis that epigenetic activation of ERVs sensitize tumors to immune therapy by recruiting
host immune cells in an immunocompetent mouse model of serous ovarian cancer. Treatment of this
model with DNMTi and HDACi results in a Type I Interferon response and increased recruitment of
(CD3+) T cells, including tumor-killing T Effector cells, to the tumor. This epigenetic therapy causes
increased activation of CD8 T cells and natural killer cells, an increase in helper T cells, and a
reduction in myeloid derived suppressor cells and macrophages. Both the immune cell activation and
the tumor burden decrease by DNMTi are dependent on activation of the Type I Interferon response
and associated with an increase in transcription of mouse ERVs. Treatment of this mouse model with
the above drug combination plus an antibody to the immune checkpoint PD-1 significantly reduces
tumor burden and increases survival. We thus define a major mechanism for how DNMTis and
HDACis may induce cancer cells to increase attraction and activation of immune cells and sensitize
patients to immunotherapy.
8
Therapeutic and Prophylactic Applications of Bacteriophages in Cancer Therapy
Biswajit Biswas, Matthew Henry, Javier Quinones, David Wolfe, and Theron Hamilton Naval Medical Research Center
Recent global surveillance indicates that bacterial pathogens are gaining Antibiotics resistances at
an alarming rate. This raises a concern about the potential of saving cancer patients from various
multidrug resistant (MDR) bacterial infections due to their reduced immune status during and after
cancer therapy. Recently, scientists have been evaluating the possibility of using bacteriophages
(phages) as therapeutic and vaccine delivery agents, but Phage therapy is also being considered
for treating cancer patients if they are infected with MDR bacteria during or after cancer therapy.
Additionally, phage can be used to develop vaccines due to the inherent properties of phages as
potent immune-stimulators. Currently, we are modifying phage heads in order to produce complex
nanoparticles which can provoke the immune system in different ways depending on the nature of
the display antigens. Importantly, these phage-based nanoparticles are non-pathogenic to humans
and the resultant vaccines do not require additional adjuvants. Phage based vaccines (PBV) can
deliver all relevant information to the immune system in order to provoke a rapid response. It is
the particulate nature of PBVs that attracts the antigen presenting cells (APC) that engulf, process,
and present phage mediated antigens through MHCs (class I and Class II pathways) and evoke
both cell mediated and humoral immunities. In addition, the immunostimulatory unmethylated
CpG motifs of the phage genome are recognized by the innate immune cells through Toll-like
receptors (TLR), which further enhance immunity. Finally, isolation of natural phages from
environmental sources is rapid, facile, and inexpensive. Therefore, developing targeted phage
cocktails for MDR-infected cancer patients is rapid. These facts encouraged us to consider phage
as prophylactic and therapeutic measures against cancer and some complications associated with
its treatment. As a preliminary attempt we have designed, developed, and produced a nanoparticle
based anticancer vaccine targeting human aspartyl (asparaginyl) b-hydroxylase (HAAH). This and
several other phage based therapeutics and prophylactic experimental results related to these
applications are presented here.
9
Cytomegalovirus-based vaccines in breast and melanoma mouse tumor models
Ann B. Hill, Michael W. Munks, Guangxu Xu, Tameka Smith, Conrad Barry, Xiaoyan Wang, Savannah Smart, Shawn Jensen, Bernard Fox, Christopher Snyder and Rosalie Sears.
Oregon Health and Science University, Portland, OR, Thomas Jefferson University, Pennsylvania, PA, and Earle A Chiles Cancer Research Center, Portland, OR.
The common herpesvirus, cytomegalovirus (CMV), is of interest as a vaccine vector because it
elicits very large, sustained T cells and antibody responses, and can be used as a vector even in
CMV seropositive individuals. In different mouse cancer models CMV vaccines have shown
therapeutic efficacy by eliciting tumor-specific CD8 T cell or antibody responses, or by an
antigen-independent impact on the tumor microenvironment when injected intratumorally. We
generated a murine (m)CMV based vaccine expressing rat HER-2/neu (CMV-neu). The vaccine
was effective prophylactically and therapeutically in the transplanted TUBO mammary tumor
model in BALB/c mice. We next tested it in a genetically engineered mouse model of mammary
cancer, in which beta lactoglobin-Cre-driven expression of neuNT and c-myc is induced in
mammary epithelium by lactation. Blg-cre/neuNT/myc mice develop mostly single mammary
tumors within several months of lactation; the tumors phenocopy human HER2 tumors, and
about 50% metastasize. A single i.p. injection of CMV-neu caused complete regression of the
majority of palpable tumors in Blg-cre/neuNT/myc mice; the CMV vector alone had no effect.
The tumors begin to regress around five days after vaccination. Immune depletion studies are
underway, but preliminary results suggest that antibody and CD4 T cells are needed for vaccine
efficacy.
0
Bacterial and eukaryotes based cancer therapy
10
Direct detection of genotoxic or pro-inflammatory bacterial genes in stool
samples: implications for colorectal cancer and inflammatory diseases
Abel Baerga-Ortiz
University of Puerto Rico Medical School
The risk of developing colorectal cancer (CRC) and inflammatory bowel diseases (IBDs) is
dependent upon a number of factors which include the gut microbiota. In recent year, advances in
automated and parallel DNA sequencing technologies have enabled a more complete
understanding of how the gut microbiota modulates the risk for intestinal disorders, resulting in
the identification of bacterial species and bacterial genes whose presence correlates with disease
state. Our group has developed a quick PCR test for the detection of some of these pro-
inflammatory or genotoxic bacterial genes directly in a stool sample. Interestingly, some of the
genes in this panel can be detected in a large percentage (>20%) of the population experimentally,
yet they only appear in a small set (1%-2%) of patient-derived sequences in the shotgun
metagenomic databases such as the Human Microbiome Project (HMP) or the European
Nucleotide Archive. Despite their low abundance in the metagenomic databases, these bacterial
pro-inflammatory and gentoxic have been shown to correlate with CRC in a case-control study
that will also be presented
11
Modulation of the Tumor Microenvironment using Synthetic BioticsTM
Adam Fisher
Synlogictx
The immunosuppressive milieu found within the tumor microenvironment (TME) has long been
understood to be a key driver of tumor initiation and progression. More recently it has been
appreciated that metabolites derived from the tryptophan and ATP/adenosine pathways are major
drivers in forming this immunosuppressive environment within the tumor. We are using
synthetic biology in combination with natural probiotics to develop engineered bacteria or
“Synthetic BioticsTM”, which are programmed with precision to correct disease-causing
metabolic dysregulation. Here we present the development of two engineered bacterial strains
that have been designed to consume either kynurenine or adenosine, two molecules known to
play central roles in promoting tumor immune tolerance. In in vitro biochemical assays, the
adenosine-consuming strain or the kynurenine-consuming strain were able to consume 180 and
80 mM adenosine or kynurenine, respectively, within 2 hours. These levels of adenosine and
kynurenine are ~100-fold and 20-fold higher respectively than found in cancer patient tumors.
For the kynurenine-consuming strain, this in vitro kynurenine consumption translated to in vivo
activity where in tumor-bearing mice, the administration of this strain led to significant decreases
in tumor kynurenine levels. Taken together these results demonstrate that engineered bacteria
can be designed to modulate the tumor microenvironmen
12
Gut microbiome analysis reveals major dysbiosis in Sickle Cell Disease patients with a prevalence of Veillonella strains
Hassan Brim, Kimberly Vilmenay, Nazli Atefi, Mohamad Daremipouran, Edward L. Lee, Sudhir
Varma, Patricia O'Neal, Hassan Ashktorab
Howard University Pathology Department & College of Medicine, Washington D.C.
Background: Sickle cell disease (SCD) is an inherited blood disorder that occurs primarily in
patients of African descent and generally associates with frequent pain crises. It has been
suggested that the gut microbiome structure and function may have a major impact on host health.
Here we used high throughput sequencing technologies to explore the gut microbiome and
specifics in SCD patients.
Aim: To characterize the gut microbiome in patients with sickle cell disease.
Materials & Methods: Stool samples from 14 controls and 14 SCD patients were used for DNA
extraction. Among the SCD patients, 7 had mild pain crises (< 3 hospitalizations/year) while 7 had
severe pain crises (≥ 3 hospitalizations/year). The 16S rRNA gene V4 variable region was PCR
amplified, purified using calibrated Ampure XP beads and used to prepare illumina DNA library.
Sequencing was performed on a MiSeq following the manufacturer’s guidelines. Sequences were
joined, and depleted of barcodes. Sequences less than 150 bp , or ambiguous base calls were then
removed. OTUs clustering was performed after the sequences were denoised, and chimeras
removed. Operational taxonomic units (OTUs) were defined by clustering at 3% divergence (97%
similarity). The final OTUs were taxonomically classified using BLASTn against a curated
database derived from RDPII and NCBI. A LeFSe analysis was used to determine differential
bacteria.
Results: A major dysbiosis was noticed in the SCD gut microbiome. Several bacterial groups have
been depleted from the SCD patients when compared with controls, including Clostridiales, a
protector against pathogens. The SCD gut microbiome has been defined by the prevalence of
Bifidobacteria, Campylobacter, Veillonella, Actinomyces, Scardovia and Atopobium. The analysis
among the two SCD groups revealed a much higher microbiota symbiosis within the SCD patients
with severe pain crises with a higher prevalence of Campylobacter strains in this group.
Conclusion: We report a major dysbiosis in SCD patients’ gut microbiota. This symbiosis is more
pronounced in patients with severe pain crises that displayed a higher prevalence of
Campylobacter. Veillonella, a normal oral and colon inhabitant, is known for its ability to form
biofilms and as a facilitator of Streptococcus strains pathogenesis. Its high prevalence in SCD
patients might exacerbate pain crises primarily due to blood vessels occlusion as a consequence of
sickle shaped blood cells. Indeed, Veillonella biofilms might block blood vessels as well and
increase Streptococcus strains virulence.
13
Reenergized Adoptive Cell Transfer (ReACT)- A Multi-pronged Strategy to Treat Solid
Tumors
Weiguo Cui PhD
Blood Research Institute, Blood Center of Wisconsin, 8727 West Watertown Plank Road, Milwaukee, WI 53213
Adoptive cell transfer (ACT) of genetically engineered T cells provides unprecedented
opportunities to treat hematological malignancies. However, due to insufficient migration and
antitumor function of transferred T cells, especially inside the immunosuppressive tumor
microenvironment (TME), the efficacy of ACT is much curtailed in treating solid tumors.
Pathogen-based cancer vaccines can break immunosuppression in TME, but are less efficient at
mobilizing tumor specific T cell response with ample magnitude to eradicate established tumors.
To overcome these challenges, we sought to combine the strengths of ACT and pathogen-based
cancer vaccines with a new strategy named Reenergized ACT (ReACT). To bridge ACT with a
pathogen, we genetically engineered tumor-specific CD8 T cells in vitro with a second T cell
receptor (TCR) that recognizes a bacterial antigen. We then transfer these dual-specific T cells in
combination with intratumoral bacteria injection to treat solid tumors in mice. The dual-specific
CD8 T cells expand robustly in response to bacteria, migrate to the very site of tumor, and confer
tumor eradication in the majority of tumor-bearing mice. ReACT demonstrates greater efficacy in
tumor control than either ACT or pathogen-based vaccine alone. The mice cured from ReACT
also develop immunological memory against subsequent tumor rechallenge. Mechanistically, we
have found that this combined approach reverts the immunosuppressive TME and recruits CD8 T
cells with enhanced killing ability to the tumors. Overall, we provide the proof-of-principle of a
promising strategy to treat various malignancies.
14
Cancer therapy in a microbial bottle: Uncorking the remarkable anti-cancer biology of
Toxoplasma gondii
Barbara A. Fox, Kiah L. Butler, Rebekah B. Guevara, and David J. Bzik
Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth,
Lebanon, New Hampshire, 03756, United States of America
Cancers emerge after the immune system fails to control and contain tumors. Multiple tumor-
specific mechanisms create tumor environments where the immune system is forced to tolerate
tumors and their cells instead of eliminating them. The goal of cancer immunotherapy is to
rescue the immune systems natural ability to eliminate tumors. Acute inflammation associated
with microbial infection has been observationally linked with the spontaneous elimination of
tumors for centuries. Here, we uncork the unique biology of the bottle-shaped protozoan parasite
Toxoplasma gondii and describe how this microbe when engineered into a safe nonreplicating
vaccine unleashes mechanisms that effectively breaks tumor control over the immune system to
stimulate potent immunity against already established cancer thereby promoting survival and
preventing cancer recurrence.
15
Developing a photosynthetic bacterial vector for intratumorial photodynamic therapy
Jill Zeilstra-Ryalls
Bowling Green State University
Many bacterial species injected into the bloodstream of mice or even administered orally
preferentially accumulate in tumors and propagate in the immunoprotective tumor
environment. In murine models, intratumorally grown bacteria inhibit tumor growth and in some
cases eradicate tumors. However, limited efficacy and concerns over safety of introducing live
bacteria into cancer patients have hindered translational applications of bacterial anticancer
therapies; at the same time, the hypoxic environment of the tumor restricts the choice of bacteria
to those species that do not require oxygen for energy production. We are investigating the
potential to use highly penetrant (but safe) near-infrared light to support intratumoral anaerobic
phototrophic growth of the nonpathogenic bacterium Rhodobacter sphaeroides. The bacteria have
been engineered to overproduce the pro-drug 5-aminolevulinic acid, which is widely used in
photodynamic therapy, and so will serve as a vehicle for targeted therapy.
16
Use of a gnotobiotic mouse model to characterize bacteria/colorectal
tumor interactions
Alexandra Proctor and Gregory J. Phillips
Department of Veterinary Microbiology, Iowa State University, Ames, IA 50010
Individuals with inflammatory bowel disease (IBD) are at a greater risk of developing colorectal
cancer (CRC). It is now evident that both IBD and CRC are associated with dysbiosis of the
gastrointestinal (GI) microbiota. Use of rodent models to study these links are complicated by the
complexity of the mammalian GI tract, however, as mice harbor hundreds of species of bacteria,
many of which are poorly characterized. Consequently, we have used altered Schaedler flora
(ASF) mice, which are colonized with only 8 bacterial species, in combination with a model of
chemically induced inflammation and mutagenesis, to better understand how members of the GI
microbiota interact with the host. Use of RNA-seq and RNA-scope technologies reveal new details
of how the microbiota responds to GI inflammation and cancer development and to identify
specific bacterial species that interact with diseased tissue. These results have implications for
developing new strategies for microbial-based CRC therapy.
17
TRAIL-armed Self-destructing Salmonella serve as “Time-Bombs” to Combat Cancer
Wei Kong Biodesign Center for Immunotherapy, Vaccines, and Virotherapy. Tempe, AZ 85287-7501
Cancer is one of the leading causes of death worldwide. In 2016, an estimated 1,685,210 new cases
of cancer will be diagnosed in the United States and 595,690 people will die from the disease due
to lack of efficient therapeutic method (NCI statistics). Bacterial cancer therapy was invented more
than 100 years ago. It is known that many passive therapeutics induce variety of side effects, but
bacteria offer unique features that can avoid these limitations. Salmonella Typhimurium (STM) is
one of the most promising bacteria using in cancer therapy. In this study, we constructed self-
destructing S. Typhimurium stains with tumor navigating feature to express TNF-related
apoptosis-inducing ligand (TRAIL) and release them inside of tumor by programmed Salmonella
cell lysis. These genetically engineered STM stains (GMS) could dramatically induce variety types
of cancer cell death in vitro. In addition, GMS stains, administrated by intra-tumor injection or
orally, induced significant tumor regression and extend survival rate of tumor bearing mice in
multiple colon cancer mouse models. Our results indicate that these TRAIL-armed Self-
destructing Salmonella can serve as an alternative novel approach for cancer therapy.
18
Presence of Multiple Responding Mechanisms in Intestinal Cancer Tissues Mediated by Microbiome Revealed by Secondary Analysis of Existing Gene Expression Data
Bi Zhao and Bin Xue
The Department of Cell Biology, Microbiology and Molecular Biology, College of Arts and Sciences, University of South Florida, Tampa, FL. USA 33620
Gene expression analysis provides valuable information on the molecular mechanisms through
which the samples undergo genetic and phenotypic changes as a respond to various internal and
external factors. By using multiple sets of existing gene expression data of intestinal cancer tissues
mediated by microbiome, differentially expressed genes (DEGs) were analyzed to: (1) characterize
the heterogeneity of tissue samples in the same control or treatment group; (2) evaluate the
influence of heterogeneity on the identification of DEGs; (3) determine different responses of
samples in the same treatment group under the same treatment; and (4) discover various
mechanisms associated with different responses. It has been found that the heterogeneity of
samples is a critical factor for tissue-level gene expression analysis.
19
Loss of Cancer Immune Privilege in Bacterial-based Therapy
*Qiuhong He, Vitaly Chibisov, Gabrielle Ramus, Huiwen Liu, Kaung-Ti
Yung, Peter Wu, Weike Lai, Xiang-jin Song, and Han H. Liu
Departments of Radiology and Bioengineering, University of Pittsburgh Cancer
Institute, University of Pittsburgh, 200 Lothrop Street, Pittsburgh, PA 15213.
We have genetically engineered the Attenuated Salmonella Typhimurium VNP20009
strain to express anti-cancer proteins TNFα and/or methioninase (METase), with or without an
imaging marker myoglobin (Mb). The therapeutic VNP20009-METase-TNFα -Mb, VNP20009
METase, VNP20009-TNFα, and the control VNP20009 and VNP20009-Mb strains were
administered i.t. or i.v. into the immunocompetent C57BL/6 mice carrying Lewis lung carcinoma
(LLC). The Salmonella Typhimurium strains carrying anti-cancer proteins have selectively
amplified in the tumor tissues, including the secondary tumor site. Rapid tumor tissue destruction
was observed by non-invasive MRI and histochemical tissue staining, accompanied with
neovascular damage and tumor cell apoptosis. Large number of macrophages was observed in
the treated LLC tumors by immunochemical staining of the tumor tissues. For tumors treated
with the control VNP20009 or VNP20009-Mb strains, the confocal microscopic images displayed
macrophage engulfment of the bacteria in the tumor tissues. This explained why the control
bacteria did not amplify in the tumor tissues and consequently caused no tumor tissue damage.
In contrast, macrophage no longer engulfed the Salmonella strains in the tumors treated with
the therapeutic VNP20009-METase-TNFα-Mb strains. The anti-cancer proteins methioninase
and/or TNFα enabled the VNP20009 strains to overcome the host immune barrier in C57BL/6J
mice for selective tumor infiltration. In addition, the LLC tumors lost immune privilege and got
destroyed upon amplification of the therapeutic VNP20009-METase-TNFα- Mb, VNP20009
METase, VNP20009-TNFα strains in the tumor tissues. The macrophages may have elicited both
innate and adaptive immune responses for tumor destruction and bacterial clearance,
respectively, in the therapeutic and control cases. Detailed molecular mechanisms merit further
investigation to induce similar anticancer effect in human cancer treatment.
20
An Archaeal Therapeutic Drug and Antigen Delivery System Employing Buoyant,
Bioengineerable Proteinaceous Nanoparticles
Shiladitya DasSarma and Priya DasSarma
University of Maryland School of Medicine, Department of Microbiology and Immunology, Institute of Marine and Environmental Technology, Baltimore, Maryland 21202
Some aquatic microbes produce large quantities of buoyant nanoparticles called gas vesicles
(or GVNPs) to promote flotation and photosynthesis. These hollow, gas-filled organelles are
bounded by an extremely stable yet biocompatible protein membrane. In the salt-loving
Archaea Halobacterium sp. NRC-1, GVNPs have been shown to be genetically engineerable
and capable of displaying foreign proteins (1). These nanoparticles are also easily purified after
hypotonic cell lysis and may serve as an ideal vehicle for therapeutic drug and antigen delivery
due to their non-toxicity and immunogenicity (1,2). Genetic analysis showed that GVNPs are
encoded by a gene cluster on Halobacterium megaplasmids, with gvpA and gvpC encoding the
major protein components. GvpC protein is bound to the exterior surface of nanoparticles and
promotes their growth and stability. We have developed a genetic system for bioengineering
the nanoparticles by insertion of foreign sequences near the C-terminal coding region of gvpC.
This expression system has permitted the display of diverse peptides and proteins on GVNPs,
including the easily detectable Gaussia princeps luciferase reporter (3). In another recent study,
GVNP display and delivery of bactericidal permeability-increasing protein (BPI) resulted in
protection of mice in a sepsis model (4). Other proteins successfully displayed by GVNPs
include antigens from the simian immunodeficiency virus (SIV), the facultative intracellular
pathogen Salmonella enterica, and parasitic protozoa Plasmodium species (1). All together, our
results have shown that the Halobacterium GVNP display and delivery system represents a
unique platform for the development of potential diagnostic assays and therapeutic
interventions.
1. DasSarma, S., and DasSarma, P. 2015. Gas Vesicle Nanoparticles for Antigen Display.
Vaccines 3:686-702.
2. Andar, A.U., Karan, R., Pecher, W.T., DasSarma, P., Hedrich, W.D., Stinchcomb, A.L.,
and DasSarma, S. 2017. Microneedle-Assisted Skin Permeation by Nontoxic Bioengineerable
Gas Vesicle Nanoparticles. Mol. Pharm. 14:953-958.
3. DasSarma, S., Karan, R., DasSarma, P., Barnes, S., Ekulona, F., and Smith, B. 2013. An
improved genetic system for bioengineering buoyant gas vesicle nanoparticles from
Haloarchaea. BMC Biotechnol. 13:112.
4. Balakrishnan, A., DasSarma, P., Bhattacharjee, O., Kim, J.M., DasSarma, S., and
Chakravortty, D. 2016. Halobacterial nano vesicles displaying murine bactericidal
permeability-increasing protein rescue mice from lethal endotoxic shock. Scientific Rep.
6:33679.
21
Bacterial Secreted Polysaccharide Toxins Bind to HP59 (Sialin) on Tumor Capillary Endothelium, Activate Complement and Recruit CD69+ Granulocytes, Disrupt Capillaries.
Roger A Laine, Department of Biological Sciences
Louisiana State University and A&M College, Baton Rouge, LA 70803. TumorEnd, LLC, Louisiana
In 1867, Busch in Germany1 observed that a sarcoma patient who developed nosocomial erysipelas
had spontaneous tumor regression. He followed up by wounding sarcoma patients, putting them in
the same hospital bed as the erysipelas patient and observed further sarcoma patients to have tumor
regression if they contracted erysipelas. Fehleisen, in 18812, identified the organism that caused
erysipelas as Streptococcus erysipelas, now called Streptococcus pyogenes, and, following Busch’s
work, injected bacterial cultures into sarcoma patients to initiate erysipelas, and infected patients had
tumor regression. Independently, William B Coley, MD, at Cornell, in 18913 discovered in hospital
records an erysipelas infected sarcoma patient who had spontaneous tumor regression, and followed
up by injecting S. erysipelas into sarcoma patients, with tumor regression in successfully infected
patients. Hemorrhage in the tumor was universal, followed by inflammation and regression. Coley
tried heat-killed bacterial cultures which had the same effect. No other organ or tissue was affected
except the tumor, and the tumors were affected whether the heat-killed cultures were injected directly
into the tumor or at distant sites. Coley began using mixed cultures of S. erysipelas and S. marscesens,
heat killed, to treat his patients. This combination became known as “Coley’s Toxin”, and was used
by a number of other physicians to significant success against sarcomas and other tumors. Beebe and
Tracy4, in New York, used Coleys toxin to treat transplantable sarcomas in dogs, also experimenting
with other bacterial extracts, and found universal tumor regression in the majority of dogs.
Hellerqvist, et al. in the 1990’s5 independently found a Group B Streptococcus (agalactica)
polysaccharide exotoxin that was associated with destruction of neocapillaries in infant human lungs
“Early Onset Disease”. Hellerqvist’s group found that tumor also attract hypoxically driven
neovasculature susceptible to GBS Toxin, which they called CM101. They showed effective tumor
therapy with CM101 in preclinical models.6 A published Phase I clinical safety trial, under IND 4578
showed 33% effectivity in Stage 4 cancer patients, including one cure7 . The receptor was later
identified by Fu, et al.8 as Sialin SLC17A5. CM101 showed safety at 10X the effective dose in a
Phase I trial in dogs, and canine cancer patients are being recruited for a Phase II trial.
1(Busch W. Aus der Sitzung der medicinischen Section vom 13 November 1867. Berl Klin Wochenschr. 1868;5:137). 2(Fehleisen F. Ueber die Zuchtung der Erysipelkokken auf kunstlichem Nahrboden und ihre Ubertragbarkeit auf den Menschen. Dtsch Med Wochenschr. 1882;8:553–554. doi: 10.1055/s¬0029¬1196806.) 3(Coley, WB, Ann. of Surg. St. Louis, 1891, xiv, p. 199; , Coley, WB, Amer. Journ. Med. Sci., Philad.1893, cv, p. 487.) 4Beebe SP, Tracy M, 1907, “The Treatment of Experimental Tumors with Bacterial Toxins”, J. Am. Med. Assoc. 18: 1493 -1498 5Sundell HW, Yan H, Carter CE, Wamil BD, Wu K, Gaddipati R, Li D, Hellerqvist CG (2000). "Isolation and identification of group B ß-hemolytic streptococcal (GBS) toxin from septic newborn infants". The Journal of Pediatrics. 137: 338–344. doi:10.1067/mpd.2000.107839. PMID 10969257 6(Thurman, GB; Russell, BA; York, GE; Wang, Y-F; Page, DL; Sundell, HW; Hellerqvist, CG (1994). "Effects of GBS toxin on long-term survival of mice bearing transplanted Madison lung tumors". J. Can. Res. Clin. Oncol. 120 (8): 479–484. doi:10.1007/BF01191801). 7(DeVore RF, Hellerqvist CG, Wakefield GB, Wamil BD, Thurman GB, Minton PA, Sundell HW, Yan HP, Carter CE, Wang YF, York GE, Zhang MH, Johnson DH (1997). "A phase I study of the antineovascularization drug CM101". J. Clin. Can. Res. 3 (3): 365–372. PMID 9815693..
22
Tumor targeting by Fusobacterium nucleatum
Jawad Abed1, Naseem Maalouf1, Lishay Parhi1, Ofer Mandelboim3 and Gilad Bachrach1
1The Institute of Dental Sciences, The Hebrew University-Hadassah School of Dental
Medicine, Jerusalem, 91120, Israel
3The Lautenberg Center of General and Tumor Immunology, The Hebrew University
Hadassah Medical School, IMRIC Jerusalem, Israel.
Colorectal adenocarcinoma (CRC) is a common tumor with high mortality rates. Recently, CRC
was found to be colonized by the oral anaerobic bacteria Fusobacterium nucleatum. F.
nucleatum accelerates tumor progression and protects the tumor from immune cells. CRC-
specific colonization by fusobacteria is mediated through the recognition of tumor displayed
Gal-GalNAc moieties by the fusobacterial Fap2 Gal-GalNAc lectin. Here, we show high Gal-
GalNAc levels in additional adenocarcinomas including those found in the stomach, prostate,
ovary, colon, uterus, pancreas, breast, lung and esophagus. This observation coincides with
recent reports that found fusobacterial DNA in some of these tumors. Given the tumorigenic
role of fusobacteria and its immune evasion properties, we suggest that fusobacterial
elimination might improve treatment outcome of the above tumors. Furthermore, if F.
nucleatum specifically homes-in to Gal-GalNAc – displaying tumors, it might be engineered as
a platform for treating CRC and the above common, lethal, adenocarcinomas.
23
Optogenetic and chemogenetic platforms for listeria-mediated intratumoral drug
delivery
R. Abrar1, C. Gravekamp2 and M. Gomelsky1
1Department of Molecular Biology, University of Wyoming, Laramie, WY
2Department of Microbiology, Albert Einstein College of Medicine, Bronx, NY
Attenuated strains of Listeria monocytogenes (Lm) accumulate and propagate in primary tumors
and metastases while being quickly cleared from healthy tissues. These strains expressing
specific tumor antigens are currently in advanced clinical trials as anticancer vaccines; however,
their curative power remains limited. We hypothesize that efficacy of the Lm-vaccines can be
significantly increased by expanding the potential of Lm as a tumor-specific payload delivery
vehicle. The payload may involve activators of innate immunity as well as tumor toxins. Since
uncontrolled production of these agents in healthy tissues infected with Lm would be
deleterious, expression needs to be inducible. We will present our efforts on engineering protein
and gene (bactofection) delivery platforms that are regulated by light or benign chemicals. Light
within the near-infrared therapeutic window penetrates deeply through mammalian tissues and
is harmless. Photoreceptors of the bacteriophytochrome class that respond to such light have
been engineered. One of the systems to be discussed involves inducible intratumoral synthesis
of c-di-GMP, the bacterial cyclic dinucleotide activator of the STING innate immunity pathway,
which results in the production of type 1 interferons. Injections of high doses of c-di-GMP in
tumor-carrying mice have been shown to induce immunogenic tumor cell death resulting in
release of tumor-associated antigens, while low c-di-GMP doses activate tumor antigen-specific
T cells. We intend to replace systemic c-di-GMP injections with localized, intratumoral c-di-
GMP production via chemically and light-controlled systems.
24
Vγ9Vδ2 T cells dominate the response to Listeria monocytogenes-based vaccines in
patients
Alejandro F. Alice1, Gwen Kramer1, Yoshinobu Koguchi1, Chris Fountain1, Rom Leidner1, Michael J. Gough1, Keith Bahjat2, and Marka R. Crittenden1,3.
1 Earle A. Chiles Research Institute, Robert W. Franz Cancer Center, Providence Portland Medical Center, 4805 NE Glisan St, Portland, OR, 97213, USA.
2 Bristol-Myers Squibb, Biologics Discovery California, Redwood City, CA, 94063, USA. 3 The Oregon Clinic, Portland, OR, 97213, USA Correspondence: [email protected]
Vector-neutralizing immunity represents a major impediment to the induction of cellular
immune responses using infectious agents as vaccine platforms. An ongoing trial
(NCT01967758) of a novel recombinant Listeria monocytogenes (Lm) –based cancer vaccine
at our institution has yielded poor tumor antigen-specific T cell immune responses. This result
is consistent with other clinical trials with Lm-based vaccines. Importantly, we observed the
marked expansion of VȖ9Vβ2 T cells in every subject (3-10 fold, 10/10 subjects). This finding
is in direct contrast to murine models, where Lm-based vaccines generate a strong αȕ T cell
response to both endogenous Lm antigens and the tumor antigen. VȖ9Vβ2 T cells recognize (E)
4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate of the non
mevalonate pathway of isoprenoid biosynthesis. Recognition of HMBPP complexed with
butyrophilin 3A1 (BTN3A1) is a property of human and non-human primate VȖ9Vβ2 T cells
that is not shared with rodent species. Therefore, traditional murine models do not take into
account the effects that these VȖ9Vβ2 vector-specific cytolytic effector cells have during Lm
immunization in humans. Following clearance of the vaccine (>5 days), VȖ9Vβ2 T cells
persisted in a constitutively activated effector-memory state (30+ days). Therefore, at the time
of secondary immunization, ~25% of the peripheral T cell repertoire is comprised of activated
Lm-specific cytolytic effector T cells. We propose that VȖ9Vβ2 T cells facilitate the rapid
clearance of Lm during secondary immunization and repeated boosts, minimizing the quantity
and quality of antigen presentation to αȕ T cells. We have designed a novel HMBPP-deficient
Lm strain that we hypothesize does not activate the dominant human Ȗβ T cell response and
may be a superior vaccine platform to generate αȕ T cell responses for human immunotherapy
approaches. These results have implications for human application of alternative Eubacteria
platforms, which similarly use the non-mevalonate isoprenoid synthesis pathway
.
25
Regulated invasion, chemotaxis, attenuation and endotoxicity of Salmonella to eliminate
tumors in tumor-bearing syngeneic mice
Shifeng Wang
University of Florida
Salmonella is a facultative anaerobe that thrives in solid tumors. We have therefore endeavored
to modify Salmonella to enhance this activity while retaining safety for healthy tissues. We first
genetically manipulated S. Typhimurium to exhibit regulated delayed in vivo attenuation and
lysis in vivo, to constitutively express invasiveness, display chemotaxis to aspartate and serine
that are produced by tumor cells and deliver TRAIL. Each of these mutational alterations
enhanced S. Typhimurium destruction of subcutaneous breast cancer tumors in syngeneic
BALB/c mice. In other studies, it was demonstrated that mutations that caused synthesis of a
truncated LPS core made Salmonella hyper invasive to colorectal tumor cells but very
susceptible to being killed by macrophages. Such mutations also caused enhanced susceptibility
of the Salmonella to being killed due to complement-mediated cytotoxicity. However, this
sensitivity to killing by host defense mechanisms also reduces the ability of the Salmonella cells
to reach and invade tumor cells. We therefore used a regulated delayed cessation in synthesis
of the LPS core by fusing the gene imparting the defect in LPS core synthesis to the arabinose
activated araC PBAD cassette. Growth of the strain in the presence of arabinose results in
complete LPS synthesis and ability after inoculation into the tumor-bearing mouse to efficiently
gain access to the tumor. LPS core synthesis ceases in vivo and the LPS Outer core and O-
antigen decreases in amount at each in vivo cell division since arabinose is not present in animal
tissues. During the course of these studies we learned that the virulence attributes of the
Salmonella parental strain very much enhanced efficacy in destroying tumors. It was also
demonstrated that inclusion of an aroA mutation also enhanced tumor cell killing. We believe
that liberation of peptidoglycan constituents during Salmonella cell lysis and which are also
elevated in aroA mutants contributes to inflammasomes to augment tumor cell killing. We are
now constructing new Salmonella combining these and additional traits to enhance tumor cell
killing and inability to be harmful for normal tissues external to the tumor
26
Contribution of Salmonella enterica serovar Typhimurium VNP20009 Chemotaxis on 4T1
Mouse Mammary Carcinoma Progression
Katherine M. Broadway, Sheryl L. Coutermarsh-Ott, Seungbeum Suh, Bahareh Behkam, Irving C. Allen, Birgit E. Scharf
Virginia Tech, 970 Washington St. SW Blacksburg, VA 24060
Attenuated bacterial strains have been investigated on the premise of selective tumor colonization
and drug delivery potential for decades. Salmonella Typhimurium VNP20009 was derived from
the parental strain 14028 through genetic modification and tumor targeting ability, being well
studied for anticancer effects in mice. In 2001 Phase 1 Clinical Trials, patients diagnosed with
melanoma were introduced with VNP20009, resulting in safe delivery of the strain and targeting
to the tumor, however no anticancer effects were observed. Recently, it was discovered that
VNP20009 contains a SNP in cheY, which encodes the chemotaxis response regulator of flagellar
motor function, rendering the strain deficient in chemotaxis. Replacement of cheY with the 14028
wild-type copy resulted in a 70% restoration of phenotype in traditional capillary assays compared
to the parental strain. We attempted to optimize the chemotactic potential of VNP20009 but were
unable without reversing the attenuated state of VNP20009.
Due to the role of chemotaxis in bacterial tumor colonization and eradication remaining unclear,
we aimed to compare VNP20009 and VNP20009 cheY+ primary tumor colonization and impact
on metastasis in an aggressive 4T1 mouse mammary carcinoma model. Based on our pilot study,
the presence of VNP20009 cheY+ significantly increased the number of metastatic foci in the lungs
and cytokine expression of cancer cells derived from the primary tumor, as compared to
VNP20009. To confirm this preliminary finding a full study was implemented, where both
intravenous and direct tumor injection routes were tested. In contrast to our pilot study, tumor
colonization and metastatic potential caused by the bacteria appear chemotaxis independent.
Moreover, mice bearing tumors exposed to Salmonella exhibited increased morbidity that was
associated with significant liver disease. Our results suggest that VNP20009 may not be safe or
efficacious when used in the context of immunocompetent animals with aggressive, metastatic
breast cancer.
27
Nanoscale Bacteria-Enabled Autonomous Drug Delivery Systems (NanoBEADS) for
Cancer Therapy
Bahareh Behkam, Associate Professor of Health Sciences Department of Mechanical
Engineering
School of Biomedical Engineering and Sciences Virginia Tech
Systemic chemotherapy is a major therapeutic approach for nearly all types and stages of cancer.
Despite significant recent progress in nanomedicine, drug delivery to solid tumors remains a
formidable challenge often associated with limited penetration of the drug in poorly vascularized
regions. In contrast, attenuated strains of tumor-targeting bacteria have been demonstrated to
have exceptionally high selectivity to cancerous tissue, a good safety profile, and effective
tumor penetration capability. However, therapeutic efficacy in immunocompetent host can be
difficult to achieve. We hypothesize that a combinatorial therapy approach based on
integrating bacteria with chemotherapeutics-loaded nanoparticles will amplify the therapeutic
potential of both modalities. Thus, we have developed a Nanoscale Bacteria-Enabled Drug
Delivery System (NanoBEADS) in which the functional capabilities of the engineered tumor-
targeting bacteria are interfaced with and augmented by chemotherapeutic-loaded
nanoparticles. We report (1) our in vitro results elucidating the mechanism of bacteria and
NanoBEADS intratumoral transport and (2) our in vivo results in mouse 4T1 breast tumor model
characterizing the intratumoral transport efficacy of NanoBEADS compared to passively
diffusing nanoparticles.
28
Induction of Autophagy and Apoptosis in Triple-Negative Breast Cancer Cells by LT-IIc, a
Type II Heat-labile Enterotoxin
Yasser Heakala, Sofia A Girald Berlingeria, c, John Hub, Matthew Federowicza, Terry D.
Connellb, Patricia Masso-Welchc
aDepartment of Pharmaceutical Sciences, D’Youville College School of Pharmacy, 320 Porter
Avenue, Buffalo, NY 14201. bDepartment of Microbiology and Immunology and the Witebsky
Center for Microbial Pathogenesis and Immunology, Jacobs School of Medicine, The University
at Buffalo, 3435 Main Street, Buffalo, NY 14214 cDepartment of Biotechnical and Clinical
Laboratory Sciences, Jacobs School of Medicine The University at Buffalo, 3435 Main Street,
Buffalo, NY 14214
Despite the recent advances [1]in breast cancer treatment, triple negative breast cancer (TNBC)
remains a serious health problem with poor prognosis and limited therapeutic options. Although
hampered by development of resistance, chemotherapy-based regimens remain the first-line
adjuvant choice for the treatment of TNBC. To combat development of resistance, it is essential
to identify agents that induce cell death by independent induction of multiple cell death pathways.
In this report, we screened cholera toxin (CT), a type I heat-labile enterotoxin (HLT) of Vibrio
cholerae, and LT-IIa and LT-IIb, type II HLT of Escherichia coli, for capacity to induce cell death
specifically in triple-negative MDA-MB-231 breast cancer cells. While CT, LT-IIa and LT-IIb had
little effect on the cells, LT-IIc reduced cell viability by ~50%, concomitant with development of
extensive intracellular vacuolation. LT-IIc-induced cytotoxicity was not observed in MCF10A, a
non-tumorigenic breast epithelial cell line, or in MCF7, an estrogen receptor-positive breast cancer
cell line. LT-IIc independently and simultaneously stimulated autophagy and apoptosis in MDA
MB-231 cells. Neither knockdown of ATG5, a key mediator of cellular autophagy, nor activation
of caspase 3/7 disrupted LT-IIc-mediated cell death. Similarly, pharmacological disruption of
autophagy using bafilomycin A1, did not interfere with LT-IIc-mediated cell death. In contrast,
treatment of MDA-MB-231 cells with rapamycin, a known inducer of autophagy, enhanced LT-
IIc mediated cell death. Collectively, these findings indicate that LT-IIc uniquely induces cell
death through the divergent induction of autophagy and autophagy-independent apoptotic
pathways.
1. Nascimento, A.V., et al., Overcoming cisplatin resistance in non-small cell lung cancer with Mad2
silencing siRNA delivered systemically using EGFR-targeted chitosan nanoparticles. Acta
Biomater, 2017. 47: p. 71-80.
29
The Streptococcal Adhesin, Scl1, Recognizes Oncofetal Fibronectin
Dudley H. McNitt1, Soo Choi1 , Flavia Squeglia2, Rita Berisio2, Slawomir Lukomski1
1Department of Microbiology, Immunology, and Cell Biology, West Virginia University School of Medicine, Morgantown, WV, USA
2Institute of Biostructures and Bioimaging, National Research Council, Naples, Italy
Group A Streptococcus (GAS) gains access to host tissue by binding cellular fibronectin isoforms
containing extra domain A (EDA/cFn). EDA/cFn is expressed during embryogenesis but not in
normal human tissue, except during wound healing and within the tumor microenvironment. The
pathogen adherence to EDA/cFn is mediated by the surface adhesin, the streptococcal collagen-
like protein 1 (Scl1). Scl1 is a homotrimeric protein that binds EDA via its N-terminal globular
sequence-variable (V) domain. The Scl1-V domain has predicted conserved structure composed
of three pairs of anti-parallel α-helices connected by flexible loops. We hypothesized that the
surface-exposed loops in the Scl1 globular domain mediate EDA recognition and binding. To test
our hypothesis, we designed, cloned, expressed in E. coli, and affinity-purified a series of the
chimeric recombinant Scl constructs by replacing the loop sequences. Circular dichroism and
rotary shadowing analyses demonstrated that chimeric proteins were not altered structurally. We
identified that the C-terminal 11 amino acids of the Scl1-V-loop segment was sufficient for rEDA
binding. Surface potential maps of the homology models of Scl1-V domains revealed conserved
negatively-charged pockets within the V-domain loop segment. Current work is focused on
immobilizing rScl1 to the surface of nanoparticles, and assessing the capacity of these
functionalized nanoparticles to adhere to rEDA-coated surfaces and to cancer-cell deposited
matrices. Our work aims to demonstrate the potential of rScl-functionalized nanoparticles as
vehicles targeting cancer tissue for diagnostic and drug delivery applications.
30
Dietary fatty acids modulate fungal-host interactions
Safanah T. Siddiqui and Melissa C. Garcia-Sherman
Department of Biology, City University of New York Brooklyn College, Brooklyn, NY 11210
Candida albicans is a major species of the fungal component of the human microbiome. It
commonly colonizes the oral, gastrointestinal and genital regions of the human body. C. albicans
becomes pathogenic when the host microbiota and immune defenses are altered causing superficial
and systemic candidiasis. The human diet can modulate immune response, inflammation levels
and the human microbiome. The average American diet is high in omega-6 fatty acids but low in
omega-3 fatty acids. Here we demonstrate that an omega-3 fatty acid, docosahexaenoic acid
(DHA), inhibits switching from yeast to the pathogenic-associated hyphal morphology. Treatment
of C. albicans with DHA mitigates biofilm formation on abiotic surfaces. Furthermore DHA also
prevents binding of C. albicans to human oral epithelial cell monolayers. Our results suggest that
DHA interferes with C. albicans morphogenesis, adhesion to biotic and abiotic surfaces. These
findings serve as a basis for future studies to determine whether DHA moderates pathogenic
interactions between C. albicans and its host.
31
Could Trypanosoma cruzi Infection Be a Good Thing in the Presence of a Tumor?
Arturo Ferreira1, Paula Abello1 , Viviana Ferreira2
1Immunology Program, Faculty of Medicine, University of Chile, Santiago, Chile; 2Department of Medical Microbiology and Immunology, College of Medical and Life
Sciences, The University of Toledo, Ohio, USA
Worldwide, 10 million people are infected with Trypanosoma cruzi, an intracellular
protozoan, but only 30% will present symptoms of Chagas’ disease. Several observations
associate T. cruzi with resistance to solid tumors: i) cancer is rare in endemic areas; ii) infected
rats resist carcinomas; iii) this parasite has tropism for tumors; iv) used as a vector for a testis
tumor antigen, it inhibits tumor growth and; v) rats treated with parasite extracts show CD4+
and CD8+ anti-tumor responses. However, no responsible parasite molecule was identified in
these studies.
We propose T. cruzi calreticulin (TcCalr, previously known as TcCRT), an ER-resident
chaperone, as a mediator of these effects, because: i) infective trypomastigotes translocate
TcCalr from the ER to the exterior, ii) non-infective T. cruzi epimastigotes fail to translocate
TcCalr; iii) T. cruzi carrying one, two or three TcCalr alleles, increasingly: express TcCalr,
resist human complement and display infectivity; iv) recombinant TcCalr (rTcCalr) inhibits:
angiogenesis in vitro, ex vivo and in ovum, proliferation and chemotaxis of human endothelial
cells in vitro; v) in molar terms, TcCalr is more anti-angiogenic than human calreticulin, vi) the
anti-angiogenic TcCalr effect is reversed by anti-rTcCalr antibodies; vii) the in vivo anti-tumor
effects of T. cruzi infection, or parasite extracts, are fully reproduced by exogenously
administrated rTcCalr and, viii). the in vivo anti-tumor effects of the infection are neutralized
by anti - rTcCalr antibodies.
Perhaps, the relevant uniqueness of TcCalr resides in its capacity to bind tumor cells and
in its amino acidic sequence differences (about 50%) from the mammal counterparts. Upon
TcCalr binding to tumor cells, infiltrating dendritic cells may present TcCalr-derived peptides
to T cells in the regional lymph nodes thus forcing an adaptive anti-tumor immune response.
Acknowledgements: A.F. laboratory is supported by grants from the CONICYT
CHILE/FONDECYT 1130099
32
Novel Treatment of Melanoma: Combined Parasite-Derived Peptide GK-1 and Anti-
Programmed Death Ligand 1 Therapy
Jesus Vera MD.
Hematology and Oncology, Mayo Clinic
background: Several studies in advanced melanoma patients suggest that combining therapies
that target tumor mechanisms of immune evasion with activation of normal immune cell
functionality, may provide optimal benefits for patients. The synthetic parasite derived GK1
peptide in combination with anti-PD-L1 showed significant longer survival (34 days) compared
to GK1 or Anti-PD-L1 alone (23-27 days) in a murine melanoma model (p<0.05). This means
an increase survival increased in 47.82% in the mice treated with GK-1 + anti-PD-L1, 21.7%
in mice treated with GK-1 alone, and 6.08% in those mice treated with anti-PD-L1 only.
Methods: To elucidate the potential mechanism by which this combination treatment exerts its
anti-melanoma effects, C57BL/6 mice were injected with B16-F10-luc2 cells and separated
according to treatments in four groups: control, GK-1, anti-PD-L1 and GK-1/anti-PDL-1.Blood
samples were collected at day 0, 14, and at euthanization or end of the experiment and monitored
for serum cytokines using mice-specific V-PLEX Pro-inflammatory Panel.
Results: On day 14, TNF-α levels in the Anti-PD-L1 and GK-1 therapy group was significantly
lower compared to control mice. At sacrifice, the combined treatment group demonstrated
significant decrease cytokine production in IL-6 and IL-10.
Conclusions: The decreased cytokine levels observed in the GK-1/anti-PD-L1 group may
partially explain the significant improved survival observed in this group. GK-1 alone is a Th1
response inductor both in vitro and in vivo as it increases IFN-Ȗ, IL-2 but not IL-4 and IL-10. It
is noteworthy that when PD-L1 signaling is reduced in T cells these cells proliferate extensively
in vitroand produce increased levels of IFN-Ȗ and IL-17, suggesting an enhanced pro-
inflammatory phenotype. It has been established that cytokines of Th2 response such as IL-4
and IL-5, IL-10 and IL-6, have tumor-promoting activity. The anti-melanoma effect of the GK
1/anti-PD-L1 combination observed in the present study could be mediated by decreasing the
pro-tumor Th2 response. These results open a new field for alteratives to potentiate the effect
of the PD-1/PD-L1 blockade pathway. Further studies to evaluate the direct translational bench-
to bed-side translational potential are being performed by our group.
32
Technologies to Support Research on Microbial based cancer therapy
33
Cloud-based Microbe Identification and Characterization Pipeline
Hsinyi Tsang
National Cancer Institute, Rockville, Maryland, U.S.A.
Recent advances in genomics have allowed researchers to sequence microbes in great depths.
As a result, biological sequence repositories, both public and private, are experiencing an
exponential growth of sequence data. This poses a major challenge in microbial analysis,
especially for bacterial and viral pathogens. In this study, we present a flexible, reproducible,
highly-scalable, and user-friendly platform for analyzing microbial sequence data on the
National Cancer Institute’s Cancer Genomics Cloud (CGC) pilots. The cloud pilots were
conceptualized in 2013 and awarded to the Broad Institute, Institute for Systems Biology and
Seven Bridges Genomics, with the goal of enhancing the utility of cancer genomics data and
facilitating analysis. Implemented in the cloud, these cloud-based platforms realize the co
localization of compute and data in a secure environment that can be easily shared with
collaborators. The tools on the CGC are run in containers and pipelines are described using
Common Workflow Language (CWL) and Workflow Description Language (WDL), which
maximizes portability and interoperability. On these three cloud platforms, we implemented
four microbe analysis pipelines, including mothur, Qiime, bioBakery and a self-contained
sequence analysis pipeline using Basic Local Alignment Search Tool (BLAST) with various
built-in graphical visualization for PCoA analysis, alpha, beta diversity and microbe abundance.
The cloud environment has proven to be a cost-effective, reproducible, and user-friendly
alternative to high-performance computing cluster, with minimal overhead and setup
requirements. These pipelines represents a necessary step in creating a publicly available and
scalable platform for microbe discovery and microbe-host interaction analysis.
34
Mass spectrometry based metabolomics platform for cancer research
Wenyun Lu, Lin Wang, Xiaoyang Su, Eileen White, Joshua D. Rabinowitz
Lewis-Sigler Institute for Integrative Genomics, Department of Chemistry, Princeton University,
NJ 08544 and Rutgers Cancer Institute of New Jersey, New Brunswick, NJ 08903
The development of metabolomics technology has led to new interests regarding metabolism in
cancer development and progression. A number of metabolite cancer biomarkers have been
discovered, as well as the identification of some novel cancer causing metabolites. We present
here a LC-MS and GC-MS based analytical platform for the comprehensive analysis of cellular
metabolites, including water-soluble metabolites, and water insoluble fatty acids, and
phospholipids. The entire workflow consists of metabolic extraction, LC-MS and GC-MS runs,
data analysis and interpretation. Standard operation procedures have been developed for the
metabolite extraction from cell culture, tissue and serum/plasma, which involve liquid extraction
using appropriate extraction solvents. Metabolites were analyzed using multiple analytical
methods on multiple dedicated instruments. Cationic water-soluble metabolites were analyzed on
a Q Exactive Plus Orbitrap mass spectrometer using hydrophilic interaction chromatography.
Anionic water-soluble metabolites were analyzed using Exactive Orbitrap mass spectrometer
coupled to reversed phase ion pairing chromatography. Fatty acids and phospholipids were
analyzed using Agilent Q-TOF instrument coupled to reversed phase chromatography. Data
analyses were performed using MAVEN program which converts the raw data into a validated
table of metabolite-specific signals. Examples will be provided to demonstrate our capability to
analyze a broad range of metabolites from real biological samples, as well as to probe the metabolic
fluxes using stable isotope tracers, all relevant to cancer metabolism and potentially microbial-
based cancer therapy.
35
Weak Signal Detection of Lung Cancer Risk
Yanming Li1, Hyokyong Grace Hong2, Yi Li1
1. Department of Biostatistics, University of Michigan, Ann Arbor, MI 48109, USA.
2. Department of Statistics and Probability, Michigan State University, East Lansing, MI
48824, USA.
Lung cancer risk is strongly associated with exposure to environmental carcinogens, in particular
to cigarette smoking. However, not all heavy smokers develop lung cancer, and lung cancer also
develops in non-smokers. Therefore, other risk factors, including genetic ones, may play a
significant role in lung cancer. A genome-wide association study (GWAS) approach has led to
valuable insights into lung cancer susceptibility genes; however, the identified loci account for an
extremely small proportion of the familial risk. Lung cancer pedigrees are rare, implying the
possibly polygenic nature of the inherited predisposition to lung cancer. Polygene refers to a gene
whose individual effect on a phenotype is too small to be observed, but which can act together
with other genes to produce observable variation. In this project, we use the term ``Marginally
Unimportant (weak) but Jointly Informative" (MUJI) variants to consider such polygenic variants.
Currently, population-based GWAS is a widely used approach in identifying genetic factors that
affect lung cancer. However, those studies (e.g. the analysis of candidate genes/polymorphisms
and genome-wide screenings) point to genetic markers individually associated with lung cancer
risk; while most population-based studies are premised on the assumption of a polygenic control
of disease risk. In this proposal, we hypothesize that lung cancers follow a polygenic model and
propose methods to identify MUJI signals searching for polygenic susceptibility to lung cancer,
which has not yet been studied formally. Given our successful preliminary experiments of the
proposed MUJI variant detection model, we hope to explain the missing heritability of lung cancer,
improving early detection thorough discovery of disease-susceptible polygenic variants.
1. Nascimento, A.V.S., A.; Bousbaa, H.; Ferreira, D.; Sarmento, B.; Amiji, M. M., Overcoming cisplatin resistance in non-small cell lung cancer with Mad2 silencing siRNA delivered systemically using EGFR-targeted chitosan nanoparticles. Acta Biomater, 2017. 47: p. 71-80.