LaBaer, Joshua

Abstract Solid tumors develop within a complex environment called the tumor microenvironment (TME), which is sculpted by the presence of other cells, such as cancer‐associated fibroblasts (CAFs) and immune cells like macrophages (Mφs). Despite the presence of immune cells, tumor cells orchestrate a tumor‐supportive environment through intricate interaction with the components of the TME. However, the specific mechanism by which this intercellular dialogue is regulated is not fully understood. To that end, the development of an organotypic 3D breast TME‐on‐a‐chip (TMEC) model, integrated with single‐cell RNA sequencing analysis, is reported to mechanistically evaluate the progression of triple‐negative breast cancer (TNBC) cells in the presence of patient‐derived CAFs and Mφs. Extensive functional assays, including invasion and morphometric characterization, reveal the synergistic influence of CAFs and Mφs on tumor cells. Furthermore, gene expression and pathway enrichment analyses identify the involvement of the KYNU gene, suggesting a potential immune evasion mechanism through the kynurenine pathway. Lastly, the pharmacological targeting of the identified pathway is investigated.

AbstractBackgroundThe role of microbes in chronic rhinosinusitis (CRS) is poorly understood. We hypothesize that analyzing prior microbial exposures via assessing microbial protein serological reactivity in CRS versus controls may offer insights for CRS etiopathogenesis.MethodsWe profiled IgG and IgA antibodies to individual microbial proteins in serum samples of CRS patients and controls using a novel high‐throughput microarray protein technology, Nucleic Acid Programmable Protein Array (NAPPA). The study was conducted on 118 subjects (39 CRS, 79 controls). A CRS‐focused NAPPA array, with 1557 potentially sero‐reactive microbial proteins elected from a pre‐screening of 6500 genes of interest was constructed. It included membrane‐associated proteins from 47 bacterial species and all proteins from 43 viral strains. Differences between CRS and controls were compared across individual antimicrobial antibodies and the species.ResultsChronic rhinosinusitis patients had significantly elevated antimicrobial antibodies compared with controls. One bacterium (Staphylococcus aureus) and three viral strains (human metapneumovirus, human herpesvirus 5, and human herpesvirus 4) were identified as sources of the proteins that showed significantly elevated sero‐reactivity in CRS patients. Within CRS, patients with polyps had elevated antibodies against S. aureus, influenza A virus (H1N1, H3N2), and rhinovirus B14. CRS patients without polyps showed more antibodies against human herpesvirus 1 and vaccinia virus WR.ConclusionsCompared with healthy controls, CRS patients’ serum samples showed significantly increased sero‐reactivity to both bacterial and viral proteins, reflecting recent or current infection or active colonization. Significantly higher antibodies against S. aureus, human metapneumovirus, human herpesvirus 5, and human herpesvirus 4 in CRS need further study.

Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell–cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.

Abstract Tumor–stroma interactions significantly influence cancer cell metastasis and disease progression. These interactions are partly comprised of the cross-talk between tumor and stromal fibroblasts, but the key molecular mechanisms within the cross-talk that govern cancer invasion are still unclear. Here, we adapted our previously developed microfluidic device as a 3D in vitro organotypic model to mechanistically study tumor–stroma interactions by mimicking the spatial organization of the tumor microenvironment on a chip. We cocultured breast cancer and patient-derived fibroblast cells in 3D tumor and stroma regions, respectively, and combined functional assessments, including cancer cell migration, with transcriptome profiling to unveil the molecular influence of tumor–stroma cross-talk on invasion. This led to the observation that cancer-associated fibroblasts (CAF) enhanced invasion in 3D by inducing expression of a novel gene of interest, glycoprotein nonmetastatic B (GPNMB), in breast cancer cells, resulting in increased migration speed. Importantly, knockdown of GPNMB blunted the influence of CAF on enhanced cancer invasion. Overall, these results demonstrate the ability of our model to recapitulate patient-specific tumor microenvironments to investigate the cellular and molecular consequences of tumor–stroma interactions. Significance: An organotypic model of tumor–stroma interactions on a microfluidic chip reveals that CAFs promote invasion by enhancing expression of GPNMB in breast cancer cells.

Abstract Purpose: With improvements in breast cancer imaging, there has been a corresponding increase in false-positives and avoidable biopsies. There is a need to better differentiate when a breast biopsy is warranted and determine appropriate follow-up. This study describes the design and clinical performance of a combinatorial proteomic biomarker assay (CPBA), Videssa Breast, in women over age 50 years. Experimental Design: A BI-RADS 3, 4, or 5 assessment was required for clinical trial enrollment. Serum was collected prior to breast biopsy and subjects were followed for 6–12 months and clinically relevant outcomes were recorded. Samples were split into training (70%) and validation (30%) cohorts with an approximate 1:4 case:control ratio in both arms. Results: A CPBA that combines biomarker data with patient clinical data was developed using a training cohort (469 women, cancer incidence: 18.5%), resulting in 94% sensitivity and 97% negative predictive value (NPV). Independent validation of the final algorithm in 194 subjects (breast cancer incidence: 19.6%) demonstrated a sensitivity of 95% and a NPV of 97%. When combined with previously published data for women under age 50, Videssa Breast achieves a comprehensive 93% sensitivity and 98% NPV in a population of women ages 25–75. Had Videssa Breast results been incorporated into the clinical workflow, approximately 45% of biopsies might have been avoided. Conclusions: Videssa Breast combines serum biomarkers with clinical patient characteristics to provide clinicians with additional information for patients with indeterminate breast imaging results, potentially reducing false-positive breast biopsies.

Abstract It is widely appreciated that early detection is the key to making inroads in the fight against cancers. Despite the availability of endoscopic screening, compliance is poor. The diagnosis of CRC is often made in advanced stages, and the consequence is a dismal 50% five year survival across all CRC. There is urgent need for biomarkers with performance that can meet the need of early diagnosis. Like most cancers, colorectal cancers (CRCs) express tumor antigens that can induce humoral immune response. The identification of the autoantibodies against tumor antigens may facilitate early diagnosis of CRC. The goal for this pilot project was to demonstrate the feasibility of discovering autoantibody biomarkers for colorectal cancer using Nucleic Acid Programmable Protein Arrays (NAPPA). Autoantibodies in serum samples collected from 40 patients with DNA mismatch repair proficient colorectal cancer who had died of their cancer within 18 months of their blood draw and from 40 age-gender matched healthy controls were profiled on NAPPA arrays with randomly selected 2000 human protein. The fact that p53, a tumor antigen that has been previously reported to be related to various cancers including CRC, was one of the top candidates based on the statistical analysis demonstrates the validity of our discovery platform. Besides p53, thirty-one candidate antigens were identified to have sensitivity greater than or equal to 25% at 95% specificity and fifty-one candidate antigens had an area under the ROC curve (AUC) equal to or greater than 0.6. Validation of the combined 83 unique genes selected based on these two different data analysis methods in an independent sample set in a blind fashion is on-going. This pilot study using late stage cancer samples demonstrates the feasibility and power of an immunoproteomics study using protein arrays. This approach may rapidly lead to identification of markers that could have relevance in monitoring disease progression as well as in early cancer detection. Citation Format: Gokhan Demirkan, Leah Soderberg, Garrick Wallstrom, Emily Szeto, Kristi Barker, Jie Wang, Joshua LaBaer, Noralane M. Lindor, Ji Qiu. Proteome scale identification of autoantibody biomarkers in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 887. doi:10.1158/1538-7445.AM2014-887