bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract IA05: Microbiota, metabolites, and antitumor immunity
Authors: McCoy Kathy D.
Published: December 2020

The intestinal microbiome heavily influences development and regulation of the immune system. The microbiome, which includes bacteria, viruses, and fungi, can also influence the initiation, development, and progression of cancer, with modulation of the immune system as one of the key pathways involved. Gut bacteria have also been found to alter the efficacy of cancer therapies, including immune checkpoint blockade therapy. These immunotherapies utilize the therapeutic potential of the immune system and have revolutionized cancer treatment. Yet this promising new strategy is not effective in all individuals (or all cancers) and has shown poor efficacy in colorectal cancer. We therefore investigated whether the intestinal microbiota could play a role in modulating immunotherapy in mouse models of colorectal cancers. We have identified specific intestinal commensal bacteria and a bacterial metabolite that control the efficacy of immune checkpoint blockade in animal models of colorectal cancer. In this session I will discuss the cellular and molecular pathways involved in this novel microbiota-microbe-immune pathway and describe the potential of bacteria-checkpoint blockade cotherapies.

Citation Format: Kathy D. McCoy. Microbiota, metabolites, and antitumor immunity [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr IA05.
Abstract IA06: Colon cancer: Microbes and communities in microbiome translation
Authors: Sears Cynthia L.
Published: December 2020

Intensive efforts are under way to understand how the colon microbiome contributes to the pathogenesis of colorectal cancer (CRC) and to determine if and how the microbiome can be utilized in the prevention and/or therapy of CRC. Both single microbes and microbial communities are proposed as contributors to human CRC. Among single microbes, attention to date has focused on enterotoxigenic Bacteroides fragilis (ETBF), (pks+) Escherichia coli, and Fusobacterium spp., although other candidates are emerging. Among communities, one aspect has been the potential contribution of mucus-invasive, dense mucosal microbial communities termed biofilms to CRC disease pathogenesis. This talk will review select concepts within ETBF’s and biofilm contribution to colon carcinogenesis. While ETBF exposure is common, the risk of colon carcinogenesis in an individual is unknown but potentially influenced by colonization state, B. fragilis toxin isotype secreted by ETBF, colon site, and the underlying genomic state of the colonic epithelial cells. With respect to colon biofilms, about 50% of human sporadic colon cancer, particularly in the right colon, display polymicrobial biofilms with marked tumor infiltration with bacteria whereas ~15% of normal colonoscopy biopsies also reveal polymicrobial biofilms. These polymicrobial biofilms are typically composed of Bacteroidetes and Lachnospiraceae with a subset of tumors, but not biopsies, also displaying Fusobacterium. Further, samples from biofilm-positive human colon cancers induce assembly of biofilms in the distal germ-free mouse colon within a week following inoculation and are procarcinogenic (JCI 129:1699, 2019). One of the common hereditary human colon cancers (familial adenomatous polyposis; APC+/−) also displays biofilms but dominated by two bacteria, (pks+) E. coli and ETBF. A mouse model of (pks+) E. coli and ETBF cocolonization exhibited accelerated tumor formation and mortality when cocolonized with the cancer-inducing bacteria, potentially by fostering increased microbial adherence, IL-17, changes in gene expression, and enhanced colonic epithelial cell DNA damage (Science 2018;359:592-97). Current directions of translational research will be presented.

Citation Format: Cynthia L. Sears. Colon cancer: Microbes and communities in microbiome translation [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr IA06.
Abstract IA07: The plasticity of the intestinal microbiota in colorectal cancer
Authors: Yang Ye , Gharaibeh Raad , Jobin Christian
Published: December 2020

Gene-environment interaction plays a key role in disease susceptibility, including colorectal cancer. Microbiota, particularly the intestinal bacteriome, plays a central role in host physiology, and the composition and activity of this consortium of microorganisms is directly influenced by known cancer risk factors such as lifestyle, diet, and inflammation. Accumulating evidence points to a role of microbiota in carcinogenesis. The mechanism by which microbiota impacts on cancer development is still unclear, but alteration in genomic stability (genetics and epigenetics) is considered a converging point. Inflammation represents a powerful condition by which microbial composition and biologic activities are altered. Using gnotobiotic technology, microbial genetic manipulation, genetically engineered mice (Apcmin/+; Il10-/-), and microbial genomics, we investigated the role of specific bacterial or consortium of bacteria in the development of colitis-associated colorectal cancer. In this lecture, I will provide evidence that specific microbial genotoxic activities originating from various strains such as Escherichia coli and Campylobacter jejuni promote development of CRC. For example, the presence of DNA-damaging toxins such as colibactin from adherent invasive Escherichia coli or cytolethal distending toxins (cdt) from Campylobacter jejuni is critical for development of colorectal cancer. I will address the relationship between inflammation and bacteria-induced carcinogenesis by studying the effect of neutralizing TNFa antibody, a biologic used to manage intestinal bowel disease, on microbial community activity. These studies represent the first step toward understanding mechanisms by which microbiota influences development of colorectal cancer, and shed light on how alteration of environmental factors modulates microbial carcinogenic potential.

Citation Format: Ye Yang, Raad Gharaibeh, Christian Jobin. The plasticity of the intestinal microbiota in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr IA07.
Abstract IA12: Fusobacterium persistence and antibiotic response in colorectal cancer
Authors: Bullman Susan
Published: December 2020

In colorectal cancer (CRC), malignant cells are surrounded by a complex microenvironment encompassing a range of nontransformed cells, but also a diverse collection of microorganisms. A growing body of evidence demonstrates the role of particular microorganisms in modulating inflammatory environments and promoting tumor growth and metastasis. Genomic analyses have consistently revealed an enrichment of the invasive bacterium Fusobacterium nucleatum in human colorectal tumors relative to noncancerous colorectal tissues. Exogenous F. nucleatum infection in animal and cellular models has also supported its cancer-promoting role. We demonstrate via microbiome analysis and microbial culture that Fusobacterium species and their co-occurring microbiota, including Bacteroides, Prevotella, and Selenomonas species, persist in liver metastasis of Fusobacterium-positive CRC. Many of the liver metastasis share the same dominant microbiome as the paired primary CRC tumors. Additionally, we have cultured Fusobacterium species from paired primary and metastatic tumors, and whole-genome sequencing analysis revealed that the same strains of Fusobacterium are present in the primary tumors and distant site metastasis, despite the tissue being resected months or even years apart. In situ hybridization analyses show that Fusobacterium is invasive in the primary tumors and distal metastasis and is associated with malignant cells. We demonstrate via microbiome analysis and microbial culture that Fusobacterium and its co-occurring microbiota also persist and remain viable in patient-derived xenografts of CRC for multiple generations in vivo. Antibiotic treatment of mice harboring these patient colon cancer xenografts led to a significant reduction in tumor Fusobacterium load, cancer cell proliferation, and overall tumor growth, suggesting that microbiome modulation could change the course of this disease. Ultimately, if a bacterial agent can directly or indirectly contribute to cancer initiation or progression, then these organisms are viable targets for cancer prevention and treatment.

Citation Format: Susan Bullman. Fusobacterium persistence and antibiotic response in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr IA12.
Abstract IA14: Characterizing the tumor microbiome and its effects on response to therapy
Authors: Straussman Ravid
Published: December 2020

The presence of bacteria in human solid tumors has been documented for over 100 years. Still, it is only in recent years that a more thorough characterization of this low-biomass microbiome has been pursued. In my lecture, I will describe our efforts to characterize the type of bacteria present across multiple human solid tumors as well as their intratumor location. I will also describe our efforts to study the effect that intratumor bacteria may have on different tumor phenotypes in general and on response to therapy in particular.

Citation Format: Ravid Straussman. Characterizing the tumor microbiome and its effects on response to therapy [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr IA14.
Abstract IA19: Fungal infection and carcinogenesis
Authors: Hu Yinling
Published: December 2020

Increased fungal infection has been reported in oral and esophageal squamous cell carcinomas (SCCs) derived from patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, immunodeficient patients, and non-autoimmune patients, as well as patients with other cancer types after radiation and chemotherapy. Thus, it is important to understand the role of increased fungal infection in tumor development. Our lab used several animal models to demonstrate that increased oral infection or colonization with Cladosporium cladosporioide (one of the most common fungi indoor and outdoor) or Candida albicans, which is one of the human commensal yeasts and opportunistic pathogens, promoted SCCs in the oral-esophageal-forestomach organs as well as distal cutaneous carcinogenesis. We revealed the mechanism underlying the relationship of increased fungal colonization with inflammatory pathways, epithelial cancer cell-oncogenic pathways, and bacteria during carcinogenesis. Those fungus-associated pathogenic phenotypes, inflammatory signaling, and epithelial oncogenic pathways observed in mice were also seen in human cancers. Based on these results in mice and human, we speculate that increased oral fungal colonization promotes human carcinogenesis.

Citation Format: Yinling Hu. Fungal infection and carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr IA19.
Abstract PR02: A mutational signature in human colorectal cancer induced by genotoxic pks+ E. coli
Authors: Puschhof Jens , Pleguezuelos-Manzano Cayetano , Huber Axel Rosendahl , van Bostel Ruben , Clevers Hans
Published: December 2020

Various species of intestinal microbiota have been associated with the development of colorectal cancer (CRC), yet a direct role of bacteria in the occurrence of oncogenic mutations has not been established. Escherichia coli can carry the pathogenicity island pks within their genomes, which encodes a set of enzymes that synthesize colibactin. This compound can alkylate nascent DNA on adenine residues, and it has been shown to induce double-strand breaks in cultured cells. We hypothesized that pks+ E. coli are carcinogenic through the mutagenic action of colibactin. To test this, we exposed human intestinal organoids for 5 months to genotoxic pks+ Escherichia coli by repeated luminal injection. Whole-genome sequencing (WGS) of clonal organoids before and after exposure revealed a distinct and reproducible mutational signature, which was completely absent in organoids that were injected with isogenic pks-deficient bacteria. This signature encompasses a single base substitution trinucleotide signature (SBS-pks) with a marked transcriptional strand bias in line with damage on adenine residues, which was accompanied by a specific indel signature (ID-pks). Moreover, a distinct preference for the wider sequence context was detected, fitting a structural model of colibactin bound to DNA. This mutational signature was detected in three cohorts containing more than 6,000 human cancer genomes, and contribution of this signature could be detected in a subset of CRC genomes and more rarely in other cancer types. Finally, we identified driver genes harboring the colibactin target motif and could attribute driver mutations from CRC genomes to the signature. Our study describes a highly specific mutational cancer signature that reflects mutagenic activity of pks+ bacteria in the intestinal lumen, which can induce oncogenic mutations and could contribute to cancer initiation.

This abstract is also being presented as Poster A02.

Citation Format: Jens Puschhof, Cayetano Pleguezuelos-Manzano, Axel Rosendahl Huber, Ruben van Bostel, Hans Clevers. A mutational signature in human colorectal cancer induced by genotoxic pks+ E. coli [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr PR02.
Abstract PR05: Elucidating role of bacteria during pancreatic ductal adenocarcinoma (PDAC)
Authors: Chandra Vidhi , Le Roux Olivereen , Riquelme Erick , Zhang Yu , Maitra Anirban , White James. R , McAllister Florencia
Published: December 2020

Long-term survival in pancreatic cancer (PC) is rare as only a minority of all resected patients survive for >5 years. Interestingly, somatic mutations do not predict long-term survivorship, highlighting the potential role of extrinsic factors. Recent studies have reported the role of the gut and tumor microbiome in pancreatic tumorigenesis and PC treatment responses. Since intratumoral bacteria have been detected in human PC tumors, possibly arising from relocation of intestinal bacteria, it is possible that intratumoral T cells get primed with microbial antigens within the tumor microenvironment. To reconstruct bacterial phylogenies, we performed prokaryotic 16S ribosomal RNA sequencing and immunoprofiling of PC tumors from “Long-Term Survivors-LTS” (>5y post-resection) and “Short-Term Survivors-STS” (<5y post-resection). Strikingly, we found that LTS from both discovery and validation cohorts had higher microbial diversity and LTS from both cohorts were enriched for a tumor microbial signature: Pseudoxanthomonas, Streptomyces, Saccharopolyspora, and Bacillus clausii. Moreover, the most overrepresented species in LTS tumors strongly correlated with enhanced immunoactivation of the tumor microenvironment, suggestive of bacterial-mediated immune cell recruitment and activation. Based on these findings, we hypothesized that gut bacteria affect PC by triggering immune responses either locally by migrating to the tumor or systemically through responses originating in the gut. To test this, we performed in vitro studies and found that single bacterial species identified from LTS tumor microbial signature can induce activation of cytotoxic T lymphocytes (CD8+ CD69+ IFNg+) only when in direct contact with antigen-presenting cells, suggesting that bacterial antigens need to be processed and presented to intratumoral T cells to induce their activation. Furthermore, through preliminary in vivo studies, we have found that orally administered single bacterial species can be delivered to the pancreas via the gut. Further work needs to determine whether single/specific LTS bacteria play a causative role in regulating tumor immunity in vivo. If the beneficial modulatory role of specific bacteria is established, then bacteriotherapy as single therapy or in combination with fecal microbial transplants could be used as innovative strategies for treatment of pancreatic cancer.

This abstract is also being presented as Poster B27.

Citation Format: Vidhi Chandra, Olivereen Le Roux, Erick Riquelme, Yu Zhang, Anirban Maitra, James. R White, Florencia McAllister. Elucidating role of bacteria during pancreatic ductal adenocarcinoma (PDAC) [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr PR05.
Abstract IA07: UVB-induced tumor heterogeneity directs immune response in melanoma
Authors: Bartok Osnat , Patkar Sushant , Cohen Sapir , Litchfield Kevin , Karathia Hiren , Lee Joo Sang , Jiménez-Sánchez Alejandro , Day Chi-Ping , Eisenbach Lea , Miller Martin , Merlino Glenn , Pikarsky Eli , Admon Arie , Swanton Charles , Ruppin Eytan , Samuels Yardena , Wolf Yochai
Published: December 2020

Little is known regarding the relationship between intra-tumor heterogeneity (ITH) and immune response in melanoma. Here, we explored the role of ITH in tumor rejection by establishing a melanoma mouse model and inducing UVB-derived mutations that increase both ITH and mutational load. This induction gives rise to highly aggressive tumors and decreased cytotoxic activity of tumor infiltrating lymphocytes (TILs). Conversely, single cell-derived melanoma clones with reduced ITH are swiftly rejected. Tumor rejection is accompanied by increased TIL reactivity and increased infiltration into the tumor core. Using phylogenetic tree analyses and mixing experiments of 20 single cell clones that lie along the phylogenetic tee we show that tumor rejection is strongly affected by number of injected clones and genetic diversity. We have, thus set up a novel, highly controlled system that enables us to study the interphase between the immune system and different layers of intra-tumor heterogeneity. Finally, the analysis of melanoma patient data identifies parallel observations, supporting the importance of ITH in determining patient survival and response to checkpoint blockade.

Citation Format: Osnat Bartok, Sushant Patkar, Sapir Cohen, Kevin Litchfield, Hiren Karathia, Joo Sang Lee, Alejandro Jiménez-Sánchez, Chi-Ping Day, Lea Eisenbach, Martin Miller, Glenn Merlino, Eli Pikarsky, Arie Admon, Charles Swanton, Eytan Ruppin, Yardena Samuels, Yochai Wolf. UVB-induced tumor heterogeneity directs immune response in melanoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA07.
Abstract IA10: Uncovering the ecology of glioblastoma with spatial digital profiling and single cell genotyping
Authors: Walentynowicz Kacper A. , Yadav Shreya , Vincentelli Cristina , Janiszewska Michalina
Published: December 2020

Glioblastoma (GBM) remains one of the biggest challenges in oncology, with median survival of ~15 months. Extreme heterogeneity of the GBM cells within each tumor results in resistance to most of the available therapies. Understanding the relationships between the heterogenous populations of GBM cells and the diverse niches they rely on is key to developing more effective treatment for this aggressive brain tumor. Analysis of the spatial localization of cells with different properties can shed light on their microenvironmental preferences, interactions and potential vulnerabilities. Therefore, we set out to profile 20 GBM cases using methods allowing for single-cell assessment of select genomic features and protein marker expression in intact tissue samples from each tumor. We constructed a tissue microarray (TMA), containing four distant areas from each tumor block to represent the intratumor heterogeneity within each tumor fragment. Based on multicolor immunofluorescent staining with markers of glial cells, immune cells and vasculature, we selected 96 regions of interest, which were then subject to digital spatial profiling for 90 protein markers associated with glioblastoma biology, neurobiology and immunology. Pearson’s correlation and Shannon index calculation based on our profiling results showed that majority of the regions cluster by their tumor of origin, however several highly diverse tumors were also identified. To link the diversity of the protein expression found with the digital spatial profiling with underlying genomic alterations, using the same TMA slide, we performed multiplexed fluorescence in situ hybridization for genes most frequently amplified in GBM. Automated quantification allowed us to collect information from 38,000 individual cells. Based on the presence or absence of the amplifications each cell was assigned a genotype. Since mutation in hTERT promoter has been reported as one of the drivers of GBM, we used STAR-FISH, an in situ single-cell mutation detection assay, to record the frequency and spatial localization of cells harboring this mutation on our TMA. Our datasets provide a unique opportunity to link single-cell genetic diversity to the phenotypic heterogeneity of the tumor cells and their microenvironment. Spatial localization of cells with distinct genotypes allows us to further dissect the interactions between the distinct subclones of GBM cells and uncover their novel dependencies.

Citation Format: Kacper A. Walentynowicz, Shreya Yadav, Cristina Vincentelli, Michalina Janiszewska. Uncovering the ecology of glioblastoma with spatial digital profiling and single cell genotyping [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA10.
Abstract IA14: Breast cancer evolution – Insights from single cell genomics
Authors: Navin Nicholas E.
Published: December 2020

In this talk I will discuss our efforts to develop and apply single cell genome sequencing methods to study premalignant breast cancer invasion, as well as our efforts to build an atlas of normal cell types in human breast tissues. To study invasion in 10 patients with ductal carcinomas in situ (DCIS) premalignancies we developed a Topographic Single Cell Sequencing (TSCS) method that has spatial resolution to measure genomic copy number profiles in patients with synchronous DCIS-IDC tissues. Our data revealed a multi-clonal invasion model, in which genome evolution occurred within the ducts leading to the generation of multiple clones that co-migrated across the basement membrane to establish the invasive carcinomas. Our group is leading a collaborative effort to establish a human breast atlas of normal tissues using single cell RNA sequencing of 30 women. These data have identified major cell types including epithelial cells (luminal HR+, luminal secretory, basal), fibroblasts, adipocytes and endothelial cells (vascular, lymphatic) as well as immune cells (lymphocytes, myeloid). For each cell type, we have identified multiple (2-5) expression states that correspond to different biological functions. Collectively these data provide a ‘human genome’ reference of normal cell types and cell states in the human breast tissues to understand how the stromal and immune microenvironment is reprogrammed in breast cancers.

Citation Format: Nicholas E. Navin. Breast cancer evolution – Insights from single cell genomics [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA14.
Abstract IA15: Inflammatory memory and tumorigenesis
Authors: Naik Shruti
Published: December 2020

Our body’s epithelia are barriers that interface with the terrestrial environment and routinely experience inflammation. Although a vast majority of these inflammatory reactions resolve, they imprint the tissue with a memory. Cells of the immune system are traditionally thought to be the bearers of this memory, allowing them to react faster to subsequent inflammatory pressures. Yet, barrier tissues are composites of epithelial, mesenchymal, nervous, vascular, and immunological networks working in unison to sustain optimal function. I discuss the enduring impact of inflammatory exposures on the skin epithelia and in particular on their long-lived stem cells, and the implications for such inflammatory training in tumor predisposition.

Citation Format: Shruti Naik. Inflammatory memory and tumorigenesis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA15.
Abstract IA20: Aberrant leukemic developmental hierarchies and MRD-specific targeting informed by single-cell biophysical and molecular profiling
Authors: Winter Peter S. , Navia Andrew , Strouf Haley , Mirza Mahnoor , Galvez-Reyes Jennyfer , Mulugeta Nolawit , Bilal Laura , Senhaji Nezha , Dennis Peter , Leahy Catharine S. , Shigemori Kay , Powers Foster , Gupta Alejandro , Calistri Nicholas , Van Scoyk Alex , Jones Kristen , Liu Huiyun , Stevenson Kristen E. , Kimmerling Robert , Stevens Mark , Weinstock David M. , Manalis Scott R. , Murakami Mark A. , Shalek Alex K.
Published: December 2020

Targeted inhibitors of essential oncogenic kinases induce high rates of clinical response but cure few patients due to the persistence of minimal residual disease (MRD). BCR-ABL mutant leukemias are a classic example of this paradigm where patients usually achieve deep remissions followed by near inevitable relapses. Multiple factors have been shown to influence how an individual patient’s leukemic cells will navigate treatment including differentiation state, mutational background, and communication with the microenvironment. Here, we use BCR-ABL-rearranged acute lymphoblastic leukemia (BCR-ABL ALL) to interrogate cell-autonomous features leading to therapeutic resistance using low-input single-cell assays. Specifically, we use a combination of primary samples and PDX models to dissect aberrant developmental hierarchies and monitor leukemic cell transcriptional and biophysical phenotype at pretreatment, MRD, and relapse. Using machine learning, we relate malignant B cells to normal development, allowing us to define leukemic developmental programs and demonstrate that these have consequences for the time to progression as well as the genetic alterations seen at relapse. Further, we determine that there are unique biophysical features tied to leukemic developmental states and that these integrative properties co-evolve with transcriptional state over the course of treatment. Finally, we demonstrate in PDX studies that it may be possible to intercept relapse by targeting specific features of MRD cells. Together, these data suggest that significant developmental hierarchies exist in ALL, tumor subpopulations can be identified directly within MRD, and their phenotypic and molecular characterization can be exploited to therapeutic effect.

Citation Format: Peter S. Winter, Andrew Navia, Haley Strouf, Mahnoor Mirza, Jennyfer Galvez-Reyes, Nolawit Mulugeta, Laura Bilal, Nezha Senhaji, Peter Dennis, Catharine S. Leahy, Kay Shigemori, Foster Powers, Alejandro Gupta, Nicholas Calistri, Alex Van Scoyk, Kristen Jones, Huiyun Liu, Kristen E. Stevenson, Robert Kimmerling, Mark Stevens, David M. Weinstock, Scott R. Manalis, Mark A. Murakami, Alex K. Shalek. Aberrant leukemic developmental hierarchies and MRD-specific targeting informed by single-cell biophysical and molecular profiling [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA20.
Abstract IA21: Stem cells play a role in human leukemia from origin to relapse
Authors: Dick John E.
Published: December 2020

Individual cancer cells exhibit functional heterogeneity of many cancer hallmarks including the capacity for sustaining long-term clonal maintenance, a stemness property involving self-renewal. Our studies established that only rare AML cells possessed such leukemia stem cell (LSC) properties and that AML is a cellular hierarchy. LSC were found to be highly relevant to human disease as gene signatures specific to LSC were much more predictive of patient response to therapy and overall survival compared to the bulk non-LSC AML cells. We have now provided insights into how LSC develop during leukemogenesis. Through sequencing of purified populations of normal blood cells, AML cells, and xenografts from paired diagnosis/relapse AML samples, we tracked the full arc of leukemia development in humans: from the cell of origin (an HSC) that acquires the first genetic mutation; to pre-leukemic clonal expansion of HSC; the generation of genetically diverse LSC; and finally to the cellular origin of relapse (rare LSC subclones) within the diagnosis sample. Similar studies have been undertaken in B-ALL where relapse-fated subclones were found within the diagnosis sample before exposure to chemotherapy. These clones possess distinct epigenetic and metabolic properties that underlie their partially resistance to chemotherapy and they possess stemness signatures that drive their ability to regenerate relapse disease. These studies resulted in identification of novel therapeutic targets of relapse-fated subclones to begin envisioning approaches to eradicate relapse before it develops. Finally, the identification of clonally expanded pre-leukemic HSPC in the diagnosis blood sample of many AML samples predicted that it should be possible to identify individuals who are at risk for progressing to AML long before AML develops. We genotyped individuals from the general population who were enrolled in the large cohort (EPIC) who eventually developed AML and compared them to the enrollment sample of others who never progressed to AML. We have found a clear signature that is able to predict with high accuracy those individuals who have age related clonal hematopoiesis who progress to AML, almost 10 years prior to AML development. This is distinct from individuals who have benign clonal hematopoiesis who never progress. These new findings offer the potential for future intervention to permit AML prevention trials.

Citation Format: John E. Dick. Stem cells play a role in human leukemia from origin to relapse [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA21.
Abstract IA24: Dissecting glioblastoma by single cell RNA-seq
Authors: Hara Toshi , Chanoch Rony , Suva Mario , Tirosh Itay
Published: December 2020

Glioblastoma is an incurable malignancy that remains poorly understood. By large-scale single cell RNA-seq and follow up experiments we found that malignant cells in glioblastoma exist in four main cellular states that recapitulate distinct steps of neural development, are influenced by the tumor microenvironment, and exhibit plasticity. Importantly, we show that while each glioblastoma contains similar cellular states, their relative frequency varies between tumors and is influenced by chromosomal aberrations as well as by the tumor microenvironment. In particular, we demonstrate that macrophages induce a transition of glioblastoma cells into mesenchymal-like (MES-like) states. This effect is mediated, both in vitro and in vivo, by macrophage-derived ligands that interact with receptors on glioblastoma cells. Finally, we show that MES-like glioblastoma states is associated with altered T cells cytotoxicity and response to immunotherapies. Overall, this work dissects the cellular diversity and interactions within the glioblastoma microenvironment, with potential implications for therapies.

Citation Format: Toshi Hara, Rony Chanoch, Mario Suva, Itay Tirosh. Dissecting glioblastoma by single cell RNA-seq [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA24.
Abstract IA26: Tumor evolution: From Darwin’s finches to breast cancer
Authors: Polyak Kornelia
Published: December 2020

Tumor evolution is driven by heterogeneity for heritable traits in combination with environmental selection forces including cancer therapies. We have been investigating intratumor cellular genetic and phenotypic heterogeneity in breast tumors and described that higher heterogeneity is associated with poor clinical outcomes. We have also developed experimental models of intratumor subclonal heterogeneity and demonstrated that more heterogeneous tumors grow faster and develop heterogeneous distant metastases. By analyzing the underlying mechanisms we defined that intratumor heterogeneity is maintained by non-cell-autonomous factors and clonal cooperation drives metastases by modulating the local and systemic microenvironments. Lastly, we have identified epigenetic enzymes as regulators of cellular transcriptomic heterogeneity and showed that by modulating the activity of these enzymes, we can decrease cellular heterogeneity and therapeutic resistance. Overall, our studies demonstrate the need to study cancer patients as a whole and to apply a combination of molecular profiling, including single cell assays, and mathematical modeling to understand how tumors evolved and to develop more effective cancer therapeutic strategies based on this knowledge.

Citation Format: Kornelia Polyak. Tumor evolution: From Darwin’s finches to breast cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA26.
Abstract PO-003: Bivalent histone modifications: Clinical targets against pancreatic cancer stem cell heterogeneity
Authors: Das Madhumita , Doke Mayur , Gonzalez Edwin , Diaz Daima , Pineda Bryan , Celli Jonathan , Ruiz Marco , Das Jayanta K.
Published: December 2020

Bivalent histone modifications, the pre-transcriptional regulators were known as the chief orchestra underlining the mechanism of development for pancreatic cancer stem cells (PANCSCs) recently. The histone bivalency methylations are related to the transcriptional activation as well as repression of the prominent histone methylations (H3K4me3 and H3K27me3) at the transcript start sequence (TSS). These epigenetic changes are recognized to initiate heterogenic cancer stem cells (CSCs) reprogramming. Recent studies showed that the heterogenic CSCs are more resistant to anti-tumor drug therapy. Our previous study showed the relationship between bivalent histone modifications with the expression of pancreatic CSCs(CD44, CD24, CD133, Ep-CAM, ALDH1A3). Our current study is focused on the importance of designing anti-cancer drugs or epigenetic small molecules against bivalent histone modifications (to inhibit the upregulation of H3K4me3 or down-regulation of H3K27me3) related the expression of heterogenic pancreatic CSCs (CD44, CD24, CD133, Ep-CAM, ALDH1A3). The Cancer Stem Cells Therapeutic Target Database Version 2.0 (CSCTTv2.0) reported the high CD133 as PAN CSCs and high CD133, CD44 showed resistance to anticancer drugs (such as docetaxel, 5-FU, irinotecan, oxaliplatin) in many cancers. The Gemcitabine, a nucleoside analog, and Metformin are only drugs used for pancreatic cancer treatment and showed resistance against heterogenic PANCSCs. The biological informatics analyses (Cytoscape database, HPIDb, Spike, BioGrid, Reactome, MBInfo) were deployed in our study to find the effect of bivalent histone modifications on the TSS of the target genes in heterogenic PANCSCs and we found the effect at TSS of target genes CD44, and EZH2/ENX1. We also found a novel small molecule KY-02111 has an effect to reduce the expression of heterogenic PANCSCs at tumorigenic in vitro 3D spheroids model of PANC-1ORGR (Gemcitabine resistant) cells. Therefore, our preliminary study may give an important clue to find further the role of KY-02111 against the heterogenic pancreatic CSCs by inhibiting the upregulation of H3K4me3 or down-regulation of H3K27me3 as a clinical target for pancreatic cancer therapy.

Citation Format: Madhumita Das, Mayur Doke, Edwin Gonzalez, Daima Diaz, Bryan Pineda, Jonathan Celli, Marco Ruiz, Jayanta K. Das. Bivalent histone modifications: Clinical targets against pancreatic cancer stem cell heterogeneity [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-003.
Abstract PO-006: Single cell transcriptomic analysis of primary head and neck cancer stem cells
Authors: Neal Molly E. Heft , The Stephanie , Owen John Henry J. , Chang Clifford , Prince Mark E.P. , Rao Arvind , Chinn Steven B.
Published: December 2020

Despite advances in treatment modalities for head and neck squamous cell carcinoma (HNSCC) survival rates remain unchanged. Tumor heterogeneity within HNSCC as well as within the tumor microenvironment (TME) has been shown to be a major driver of treatment failure and poor outcomes. A key component of the TME and a potential key to deciphering tumor heterogeneity are cancer stem cells. While numerous studies have demonstrated the tumorigenicity and malignant potential of these cells, few studies have investigated their expression profiles. The aim of this study is to leverage single cell technology to investigate the expression profile of primary HNSCC cancer stem cells. We performed single cell sequencing of five primary HPV-negative head and neck cancers and two HNSCC cell lines that were presorted into the CSC (CD44high/ALDH+) and non-CSC (CD44low/ALDH-) phenotype. We are able to characterize an expression profile of the CSC TME niche and a novel CSC expression profile (DCN, C110rf96, KLF4, MEG3 and NR4A1) relative to non-CSC. We confirm this CSC profile is conserved across all five primary tumors. Together these data provide some of the first insights into CSC transcriptome, further define the CSC-TME niche, and identify conserved targets within HNSCC CSC as a means to decipher the challenges inherent to tumor heterogeneity.

Citation Format: Molly E. Heft Neal, Stephanie The, John Henry J. Owen, Clifford Chang, Mark E.P. Prince, Arvind Rao, Steven B. Chinn. Single cell transcriptomic analysis of primary head and neck cancer stem cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-006.
Abstract PO-012: Metastatic bottleneck in osteosarcoma selects for certain clonal populations, while metastatic lesions exhibit diverse phenotypes
Authors: Rajan Sanjana , Cam Maren , Gross Amy , Reinecke James , Taslim Cenny , Roberts Ryan
Published: December 2020

This study focuses on identifying if the metastatic niche in osteosarcoma (OS) enriches for cells that have a survival advantage either from pre-defined genetic programs or adaptive responses to environmental cues. OS patients who develop metastases experience a dismal survival rate that is lower than twenty percent, a statistic that remains unchanged despite four decades of efforts to improve these outcomes. While therapies that treat metastasis could dramatically improve outcomes, we understand too little about the mechanisms of lung colonization to propose any rational intervention. To simultaneously evaluate changes in clonal diversity and phenotype during the metastatic bottleneck, cell by cell, we combined prospective lineage tracing with single cell transcriptomics in several murine models of metastatic OS. Single cell suspensions of metastatic tumor tissues were processed for single-cell RNA sequencing (scRNA-Seq). For lineage tracing studies, OS cells were virally-transduced to contain a high diversity, heritable and scRNA-Seq compatible lineage tag before inoculating mice. Single-cell libraries were processed bioinformatically to overlay phenotype and lineage information for each cell. Our findings support the following conclusions: First, that the metastatic bottleneck selects for certain clones with increased colonizing capacity, but this selection does not narrow the overall phenotypic diversity of those cells–clones of many different phenotypes can be found within the metastases. Second, the metastatic lung enriches for certain features that are conserved across different cell line and PDX models of metastatic OS. Gene set enrichment analysis of genes enriched within the surviving clones identified novel activated signaling pathways and upstream regulators that appear critical for metastatic progression. This signature includes activated IL6, TGF-β, PDGF signaling, response to a previously unidentified upstream regulator, IL-1β, and the acquisition of enhanced glycolytic activity. Whether each lesion becomes colonized by a phenotypically-distinct clone giving rise to phenotypically-diverse lesions or whether cooperation between phenotypically-distinct clones is required to effect colonization remains a point of active study. In summary, metastatic colonization of the lung by OS involves the selection of phenotypically-diverse tumor cell clones that share characteristics that engender fitness within the lung microenvironment.

Citation Format: Sanjana Rajan, Maren Cam, Amy Gross, James Reinecke, Cenny Taslim, Ryan Roberts. Metastatic bottleneck in osteosarcoma selects for certain clonal populations, while metastatic lesions exhibit diverse phenotypes [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-012.
Abstract PO-014: Harnessing the heterogeneity of circulating hybrid cells in pancreatic adenocarcinoma
Authors: Walker Brett Scott , Sengupta Sidharth , Parappilly Michael , Ors Aysegul , Fischer Jared , Sutton Thomas , Sheppard Brett , Mayo Skye , Wong Melissa
Published: December 2020

Pancreatic adenocarcinoma (PDAC) remains one of the most lethal malignancies in the United States and tumor heterogeneity has major implications on the effectiveness of systemic therapies. Comprehensive tumor analysis at the single cell level offers an in-depth examination of neoplastic cells with metastatic potential and supports development of individualized treatment strategies to ultimately improve survival. Hybrid cells are a novel cell population derived from immune-cancer cell fusion within solid tumors and disseminate into peripheral blood, where they can be detected as circulating hybrid cells (CHCs) based on their dual expression of tumor and immune cell epitopes. Compared to conventionally-defined circulating tumor cells (CTCs), CHCs are found in patients at higher levels which allows for more robust phenotypic analyses of tumor cell features and collection for downstream multi-omic profiling. To investigate heterogeneity among disseminated CHCs in patients with PDAC, we assessed for tumor cell features using immunohistochemical analyses of single cells from peripheral blood mononuclear cell smears and by single cell RNA sequencing (scRNA-seq) from FACS-isolated cells. Downstream image analyses was performed Zeiss Zen blue software and scRNA-seq was analyzed using the Seurat analysis package. We determined that PDAC patients harbored heterogeneous populations of CHCs with differential protein expression that mirrored expression in corresponding primary tumors. Further, scRNA-seq analyses identified CHC populations with varying degrees of retained tumor identity, as well as subsets derived from different immune cell lineages. Unsupervised clustering of CHCs with tumor-dominate phenotypes demonstrated high differential expression of cancer related genes with known implications in PDAC tumors and treatment resistant pathways. Inference copy number variation analysis revealed chromosomal amplifications of 1q and 19q along with deletions of 6p, 12q, and 22q, characteristic of PDAC tumors. Additionally, a myriad of KRAS mutations were identified in patient CHCs but absent in normal leukocytes. As the product of tumor and immune cell fusion, CHCs provide a rich source for assessing PDAC heterogeneity through genetic and protein expression analyses. Ultimately, CHCs have great potential as a liquid biopsy in pancreatic cancer which could be harnessed to guide therapy.

Citation Format: Brett Scott Walker, Sidharth Sengupta, Michael Parappilly, Aysegul Ors, Jared Fischer, Thomas Sutton, Brett Sheppard, Skye Mayo, Melissa Wong. Harnessing the heterogeneity of circulating hybrid cells in pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-014.
Abstract PO-020: Inferring copy number substructure from single-cell transcriptomics in human tumors with CopyKat
Authors: Gao Ruli , Bai Shanshan , Ying Henderson , Lin Yiyun , Seth Tapsi , Hu Min , Sei Emi , Davis Alexander , Wang Fang , Wang Jennifer Rui , Chen Ken , Moulder Stacey , Lai Stephen , Navin Nicholas
Published: December 2020

High-throughput single cell transcriptomics analysis is widely used to study human tumors, however a major challenge is distinguishing the stromal cells from the malignant cancer cells, as well as clonal substructure within tumors. To address this challenge, we developed an integrative Bayesian segmentation approach, COPYKAT to estimate genomic copy numbers at 5MB resolution from high-throughput single cell RNA-seq data. We applied COPYKAT to 39,709 single cells from 16 tumors across 4 cancer types, including premalignant and triple-negative breast cancers, pancreatic ductal adenocarcinomas, and anaplastic thyroid cancer. From these data we could accurately (98%) classify tumor cells from stromal cells. In three TNBC tumors COPYKAT resolved multiple clonal subpopulations of genotypes that differed in expression of breast cancer genes and enrichment of cancer hallmarks including EMT and hypoxia. These data show that COPYKAT can accurately resolve clonal copy number substructure in tumors and classify tumor and normal cells in a variety of human cancers.

Citation Format: Ruli Gao, Shanshan Bai, Henderson Ying, Yiyun Lin, Tapsi Seth, Min Hu, Emi Sei, Alexander Davis, Fang Wang, Jennifer Rui Wang, Ken Chen, Stacey Moulder, Stephen Lai, Nicholas Navin. Inferring copy number substructure from single-cell transcriptomics in human tumors with CopyKat [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-020.