Single-cell catalogue of PM tumor and PBMC samples

Unbiased discovery of PM cancer programs and association with patient survival

Gene signatures for all 54 de novo discovered PM cNMF modules

SCENIC-predicted regulon targets of ETS1 and MEF2C in PM endothelial cells

De novo markers discovered for each major cell type annotation in tumor samples

NK cell IC blockade targeting NKG2A as a novel therapeutic strategy in PM

Molecular dissection of T cell programs and IC molecules shows association with scS-score

De novo markers discovered for each detailed cell type annotation in the peripheral blood samples

De novo markers discovered for each major cell type annotation in the peripheral blood samples

Macrophages in scS-high PM express CXCL9/10/11 and likely contribute to T-cell infiltration

Fetal-like, cancer-specific PLVAP+ endothelial cells associate with angiogenesis

Immunotherapies have shown great promise in pleural mesothelioma (PM), yet most patients still do not achieve significant clinical response, highlighting the importance of improving the understanding of the tumor microenvironment (TME). Here, we utilized high-throughput, single-cell RNA sequencing (scRNA-seq) to de novo identify 54 expression programs and construct a comprehensive cellular catalog of the PM TME. We found four cancer-intrinsic programs associated with poor disease outcome and a novel fetal-like, endothelial cell population that likely responds to VEGF signaling and promotes angiogenesis. Across cellular compartments, we observe substantial difference in the TME associated with a cancer-intrinsic sarcomatoid signature, including enrichment in fetal-like endothelial cells, CXCL9+ macrophages, and cytotoxic, exhausted, and regulatory T cells, which we validated using imaging and bulk deconvolution analyses on independent cohorts. Finally, we show, both computationally and experimentally, that NKG2A:HLA-E interaction between NK and tumor cells represents an important new therapeutic axis in PM, especially for epithelioid cases. Significance: This manuscript presents the first single-cell RNA sequencing atlas of PM tumor microenvironment. Findings of translational relevance, validated experimentally and using independent bulk cohorts, include identification of gene programs predictive of survival, a fetal-like endothelial cell population, and NKG2A blockade as a promising new immunotherapeutic intervention in PM.

While CRISPR screens are helping uncover genes regulating many cell-intrinsic processes, existing approaches are suboptimal for identifying extracellular gene functions, particularly in the tissue context. Here, we developed an approach for spatial functional genomics called Perturb-map. We applied Perturb-map to knock out dozens of genes in parallel in a mouse model of lung cancer and simultaneously assessed how each knockout influenced tumor growth, histopathology, and immune composition. Moreover, we paired Perturb-map and spatial transcriptomics for unbiased analysis of CRISPR-edited tumors. We found that in Tgfbr2 knockout tumors, the tumor microenvironment (TME) was converted to a fibro-mucinous state, and T cells excluded, concomitant with upregulated TGFβ and TGFβ-mediated fibroblast activation, indicating that TGFβ-receptor loss on cancer cells increased TGFβ bioavailability and its immunosuppressive effects on the TME. These studies establish Perturb-map for functional genomics within the tissue at single-cell resolution with spatial architecture preserved and provide insight into how TGFβ responsiveness of cancer cells can affect the TME.

Background Immune checkpoint blockade (ICB) is increasingly becoming the standard of care for many tumor types. Yet, useful predictive biomarkers for benefit from ICB are still lacking. Emerging literature has suggested the importance of intra-tumoral B cells in response to ICB; however, these studies fail to address the heterogeneity amongst B cells responses.1-4 Here, we describe the largest single-cell RNA sequencing (scRNAseq) data set of B cells in treatment-naïve and ICB-treated hepatocellular carcinoma (HCC) patients and find specific B cell phenotypes that are highly associated with response to ICB. Methods A cohort of 44 patients with early-stage HCC underwent surgical resection, of which, 26 patients received neoadjuvant anti-PD-1 treatment. Pre-and post-treatment blood and representative samples from resected tumor and adjacent uninvolved liver were collected fresh, and 10x scRNAseq was performed on the immune cell compartment. ELISA was performed on plasma from each patient to detect antibodies against a panel of 26 tumor antigens. Results We identified three major subsets of B cells: naïve, memory (Bmems), and plasma cells (PCs), and identified different clusters within each subset with distinct transcriptional signatures. Bmem and PC clusters demonstrated differential expression of the immunoglobulin isotypes and subclasses, and we hypothesized that the B cell response in ICB responders might be skewed towards an IgG1 phenotype. Indeed, we observed that non-responders had a higher proportion of Bmems from clusters with the lowest IgG1 expression, while responders had an enrichment in Bmems originating from the cluster with the highest IgG1 expression. More strikingly, while we found an overall increase in PCs in responders compared to non-responders, there was a significantly higher enrichment of IgG1-high PC clusters in ICB responders compared to non-responders. Single-cell BCRseq on five patients (2 responders and 3 non-responders) revealed that the tumor-derived Bmems and PCs in responders were more clonally expanded than those from non-responders. Furthermore, we observed that while the clonally expanded B cells in non-responders were dominated by IgA or IgM isotypes, the clonally expanded B cells in responders were exclusively IgG1 or IgG3. Finally, we detected peripheral IgG antibodies against at least one tumor-antigen in our panel in 63% of the responders—which were mostly dominated by IgG1—whereas only 22% of non-responders had detectable IgG titers against any of the tumor antigens. Conclusions HCC responders to ICB had B cell responses skewed towards an IgG1 phenotype, and the presence of anti-tumor IgG1 antibodies correlated with ICB response. Acknowledgements This study was funded by Regeneron Pharmaceuticals. We thank all of the patients and their families for their involvement in this study. Trial Registration NCT03916627 References Cabrita R, Lauss M, Sanna A, Donia M, Skaarup Larsen M, Mitra S, Johansson I, Phung B, Harbst K, Vallon-Christersson J, van Schoiack A, Lövgren K, Warren S, Jirström K, Olsson H, Pietras K, Ingvar C, Isaksson K, Schadendorf D, Schmidt H, Bastholt L, Carneiro A, Wargo JA, Svane I, Jönsson G. Tertiary lymphoid structures improve immunotherapy and survival in melanoma. Nature. 2020;577(7791):561–565. Griss J, Bauer W, Wagner C, Simon M, Chen M, Grabmeier-Pfistershammer K, Maurer-Granofszky M, Roka F, Penz T, Bock C, Zhang G, Herlyn M, Glatz K, Läubli H, Mertz K, Petzelbauer P, Wiesner T, Hartl M, Pickl W, Somasundaram R, Steinberger P, Wagner S. B cells sustain inflammation and predict response to immune checkpoint blockade in human melanoma. Nat Commun. 2019;10(1):4186 Helmink B, Reddy S, Gao J, Zhang S, Basar R, Thakur R, Yizhak K, Sade-Feldman M, Blando J, Han G, Gopalakrishnan V, Xi Y, Zhao H, Amaria R, Tawbi H, Cogdill AP, Liu W, LeBleu V, Kugeratski F, Patel S, Davies M, Hwu P, Lee J, Gershenwald J, Lucci A, Arora R, Woodman S, Keung E, Gaudreau P, Reuben A, Spencer C, Burton E, Haydu L, Lazar AJ, Zapassodi R, Hudgens C, Ledesma D, Ong S, Bailey M, Warren S, Rao D, Krijgsman O, Rozeman E, Peeper D, Blank C, Schumacher T, Butterfield L, Zelazowska M, McBride K, Kalluri R, Allison J, Petitprez F, Fridman W, Sautès-Fridman C, Hacohen N, Rezvani K, Sharma P, Tetzlaff M, Wang L, Wargo J. B cells and tertiary lymphoid structures promote immunotherapy response. Nature. 2020;577(7791):549–555. Petitprez F, de Reyniès A, Keung E, Chen TW, Sun C, Calderaro J, Jeng Y, Hsiao LP, Lacroix L, Bougoüin A, Moreira M, Lacroix G, Natario I, Adam J, Lucchesi C, Laizet YH, Toulmonde M, Burgess M, Bolejack V, Reinke D, Wani K, Wang W, Lazar A, Roland C, Wargo J, Italiano A, Sautès-Fridman C, Tawbi H, Fridman W. B cells are associated with survival and immunotherapy response in sarcoma. Nature. 2020;577(7791):556–560. Ethics Approval This study was approved by Icahn School of Medicine at Mount Sinai9s institutional review board, approval numbers 19-0246 and 19-06-061-05. All study participants gave informed consent before taking part in this study.

Background: Malignant pleural mesothelioma (MPM) is usually fatal, though multimodality therapy— now including immunotherapy— has improved survival. Recurrence after surgery is close to 100%, even with adjuvant chemotherapy and radiation. Our collaborators have performed deep immunophenotyping of treatment-naïve MPM lesions using mass cytometry (CyTOF) and single-cell RNA sequencing (scRNAseq) to define the tumor microenvironment. A population of rare CD141+ dendritic cells (DC1) is disproportionately represented in some MPM lesions analyzed. These DC1 cells— which express high levels of Toll-like receptor 3 (TLR3)— are among the most potent cross-presenters of antigen and are key to priming anti-tumor CD4+ and CD8+ T cell responses. Polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (poly-ICLC), is a double-stranded RNA host-targeted therapeutic viral-mimic. Poly-ICLC activates multiple innate immune receptors including TLR3 and melanoma differentiation-associated gene 5 (MDA5), leading to cross-presentation of antigen to T cells and induction of strong Th1 response. We hypothesize that injection of poly-ICLC prior to surgical resection may activate intratumoral (IT) DC1s, increase tumor antigen presentation to cytotoxic T cells, and induce tumor-specific immune surveillance. Methods: This is a phase I/Ib study to evaluate the safety of IT poly-ICLC prior to surgical resection for patients with MPM (NCT04525859). The primary endpoint is safety as assessed by frequency and severity of toxicities by CTCAE 5.0. Secondary endpoints are objective response as measured by RECIST 1.1 and recurrence free survival measured from the time of first poly-ICLC injection. Exploratory endpoints include evaluation of circulating immune cells (including regulatory T cells and NK cells), evaluation of immune cell infiltration in pre-injection tumor biopsy and surgically resected tissue, as well as characterization of immune parameters such as local B cell specificity. The protocol features a Simon’s two-stage design, with six patients enrolled in a phase I safety cohort, proceeding to a phase Ib expansion cohort (additional 13 patients) if no more than 1 dose limiting toxicity occurs. Eligible patients must have MPM deemed operable by the treating thoracic surgeon. Eligible subjects may not have uncontrolled immunocompromised states or autoimmune disorders. After enrollment, patients undergo biopsies at which time 2mg poly-ICLC is injected across two sites in the tumor. Patients then undergo resection of the tumor (pleurectomy/decortication or extra pleural pneumonectomy per standard of care) at day 21+/- 7 after poly-ICLC injection. Blood is drawn at three points (prior to poly-ICLC injection, at time of surgery, and at a post-operative visit) for immune profiling. At the time of submission six patients have been treated and phase Ib accrual is continuing as planned. Interim analysis of phase I safety and exploratory endpoints will be reported in late 2022. Citation Format: Bailey G. Fitzgerald, Thomas U. Marron, Robert Sweeney, Jorge Gomez, Nicole Hall, Daniel O'Grady, Christian Rolfo, Raj Veluswamy, Deborah Doroshow, John Mandeli, David Yankelevitz, Nina Bhardwaj, Sacha Gnjatic, Fred R. Hirsch, Miriam Merad, Alexander Tsankov, Raja Flores, Andrea Wolf. A phase I/Ib trial of intratumoral Poly-ICLC in resectable malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT205.

Disease states and cellular compartments can display a remarkable amount of heterogeneity, and truly appreciating this heterogeneity requires the ability to detect and probe each subpopulation present. A myriad of recent single-cell assays has allowed for in-depth analysis of these diverse cellular populations; however, fully understanding the interplay between each cell type requires knowledge not only of their mere presence but also of their spatial organization and their relation one to the other. Immunohistochemistry allows for the visualization of cells and tissue; however, standard techniques only allow for the use of very few probes on a single specimen, not allowing for in-depth analysis of complex cellular heterogeneity. A number of multiplex imaging techniques, such as immunofluorescence and multiplex immunohistochemistry, have been proposed to allow probing more cellular markers at once; however, many of these techniques still have their limitations. The use of fluorescent markers has an inherent limitation to the number of probes that can be simultaneously used due to spectral overlap. Moreover, other proposed multiplex IHC methods are time-consuming and require expensive reagents. Still, many of the methods rely on frozen tissue, which deviates from standards in human pathological evaluation. Here, we describe a multiplex IHC technique, staining for consecutive markers on a single slide, which utilizes similar steps and similar reagents as standard IHC, thus making it possible for any lab with standard IHC capabilities to perform this useful procedure. This method has been validated and confirmed that consecutive markers can be stained without the risk of cross-reactivity between staining cycles. Furthermore, we have validated that this technique does not lead to decreased antigenicity of subsequent epitopes probed, nor does it lead to steric hindrance.

In this research, we present a new transcriptome assembly of the Santa Cruz wild banana slug (Ariolimax dolichophallus), the species most important to redwood ecology. Its functional annotation, the first of its kind, will be provided freely. RNA‐Seq with Illumina HiSeq technology was used to analyze samples extracted from slug. We used Trinity and Trinotate for transcriptome assembly and annotation. This assembly and annotation greatly enriches the studies of the banana slug, given its is the first study of banana slug exonic expression. This large‐scale gene discovery gives the broadest depth yet to the annotated transcriptome of this important species and should be of value to ongoing genomic studies of banana slug behavior with an emphasis on their roles in redwood ecology and seed dispersion.

Many high‐school science education programs do not train students to be independent researchers that make novel contributions to the scientific community, which is arguably the primary goal of a scientist. There are many possible explanations for this paradox. Due to high student/teacher ratios, teachers are unable to give the individualized attention necessary to cultivate a scientist from a student. Class schedule, student motivation, and diverse intellectual support are also huge barriers. As a result, most high‐school programs struggle to establish a system that allows for university and graduate‐level laboratory education of a significant number of students.Here, we report a curriculum that trains students to design, conduct, manage, lead, and present novel research projects, but also a program that is self‐sustaining and scalable. The system produces a three‐tiered system of research students, each with an increasing set of laboratory and troubleshooting, goal‐setting, and project management skills. This management system is scaffolded. It can be completed at different speeds. These teams design projects that are novel and real. Trained teams come together and systematically create projects that can not only sustain themselves and pursue novel intellectual discovery, but also projects that are able to expand and adapt through multiple years and to a changing number of students, independent of staffing.Support or Funding InformationThis work was supported by The Nueva School.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Major Depressive Disorder (MDD) is the leading cause of disability worldwide. One of MDD's most troubling symptoms is the loss of reward firing, or anhedonia. Current publications indicate that MTCH‐2, a gene associated with mitochondrial transport, is related to a decrease in firing rates in the hippocampus of mice. Additionally, a GWAS reported a link between MTCH‐2 and neuroticism in humans. Consequently, we hypothesize that a MTCH‐2 related decrease in firing rates in the nucleus accumbens could potentially play a role in the development of depressive symptoms.The C. Elegans MTCH‐1 protein shows a 87% homology to the human MTCH‐2 protein. Here, we examine the relationship between the MTCH‐1 gene and C. Elegans reward firing. Because C. Elegans reward firing levels have been associated with changes in speed change fluidity in past studies, we measure C. Elegans motility as a proxy for reward firing. In this study, we compare motility and gene expression in MTCH‐1 deficient worms and wt worms and further examine MTCH‐1 knockdown to control motility. Together, these data points may indicate a role of MTCH‐2 in depression.Support or Funding InformationThe Nueva SchoolThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.