Dynamic thresholding and tissue dissociation optimization for CITE-seq identifies differential surface protein abundance in metastatic melanoma.

Multi-omics profiling by CITE-seq bridges the RNA-protein gap in single-cell analysis but has been largely applied to liquid biopsies. Applying CITE-seq to clinically relevant solid biopsies to characterize healthy tissue and the tumor microenvironment is an essential next step in single-cell translational studies. In this study, gating of cell populations based on their transcriptome signatures for use in cell type-specific ridge plots allowed identification of positive antibody signals and setting of manual thresholds. Next, we compare five skin dissociation protocols by taking into account dissociation efficiency, captured cell type heterogeneity and recovered surface proteome. To assess the effect of enzymatic digestion on transcriptome and epitope expression in immune cell populations, we analyze peripheral blood mononuclear cells (PBMCs) with and without dissociation. To further assess the RNA-protein gap, RNA-protein we perform codetection and correlation analyses on thresholded protein values. Finally, in a proof-of-concept study, using protein abundance analysis on selected surface markers in a cohort of healthy skin, primary, and metastatic melanoma we identify CD56 surface marker expression on metastatic melanoma cells, which was further confirmed by multiplex immunohistochemistry. This work provides practical guidelines for processing and analysis of clinically relevant solid tissue biopsies for biomarker discovery.

Acquired resistance to anti-PD1 therapy in patients with NSCLC associates with immunosuppressive T cell phenotype.

Immune checkpoint inhibitor treatment has the potential to prolong survival in non-small cell lung cancer (NSCLC), however, some of the patients develop resistance following initial response. Here, we analyze the immune phenotype of matching tumor samples from a cohort of NSCLC patients showing good initial response to immune checkpoint inhibitors, followed by acquired resistance at later time points. By using imaging mass cytometry and whole exome and RNA sequencing, we detect two patterns of resistance¨: One group of patients is characterized by reduced numbers of tumor-infiltrating CD8+ T cells and reduced expression of PD-L1 after development of resistance, whereas the other group shows high CD8+ T cell infiltration and high expression of PD-L1 in addition to markedly elevated expression of other immune-inhibitory molecules. In two cases, we detect downregulation of type I and II IFN pathways following progression to resistance, which could lead to an impaired anti-tumor immune response. This study thus captures the development of immune checkpoint inhibitor resistance as it progresses and deepens our mechanistic understanding of immunotherapy response in NSCLC.

Genomic and Transcriptomic Analyses of Malignant Pleural Mesothelioma (MPM) Samples Reveal Crucial Insights for Preclinical Testing.

Cell lines are extensively used to study cancer biology. However, the use of highly passaged commercial cell lines has to be questioned, as they do not closely resemble the originating tumor. To understand the reliability of preclinical models for Malignant pleural mesothelioma (MPM) studies, we have performed whole transcriptome and whole exome analyses of fresh frozen MPM tumors and compared them to cell lines generated from these tumors, as well as commercial cell lines and a preclinical MPM mouse model. Patient-derived cell lines were generated from digested fresh tumors and whole exome sequencing was performed on DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor samples, corresponding patient-derived cell lines, and normal tissue. RNA sequencing libraries were prepared from 10 fresh frozen tumor samples, the 10 corresponding patient-derived cell lines, and 7 commercial cell lines. Our results identified alterations in tumor suppressor genes such as FBXW7, CDKN2A, CDKN2B, and MTAP, all known to drive MPM tumorigenesis. Patient-derived cell lines correlate to a high degree with their originating tumor. Gene expressions involved in multiple pathways such as EMT, apoptosis, myogenesis, and angiogenesis are upregulated in tumor samples when compared to patient-derived cell lines; however, they are downregulated in commercial cell lines compared to patient-derived cell lines, indicating significant differences between the two model systems. Our results show that the genome and transcriptome of tumors correlate to a higher degree with patient-derived cell lines rather than commercial cell lines. These results are of major relevance for the scientific community in regard to using cell lines as an appropriate model, resembling the pathway of interest to avoid misleading results for clinical applications.

gExcite: a start-to-end framework for single-cell gene expression, hashing, and antibody analysis.

SUMMARY: Recently, CITE-seq emerged as a multimodal single-cell technology capturing gene expression and surface protein information from the same single cells, which allows unprecedented insights into disease mechanisms and heterogeneity, as well as immune cell profiling. Multiple single-cell profiling methods exist, but they are typically focused on either gene expression or antibody analysis, not their combination. Moreover, existing software suites are not easily scalable to a multitude of samples. To this end, we designed gExcite, a start-to-end workflow that provides both gene and antibody expression analysis, as well as hashing deconvolution. Embedded in the Snakemake workflow manager, gExcite facilitates reproducible and scalable analyses. We showcase the output of gExcite on a study of different dissociation protocols on PBMC samples. AVAILABILITY AND IMPLEMENTATION: gExcite is open source available on github at https://github.com/ETH-NEXUS/gExcite_pipeline. The software is distributed under the GNU General Public License 3 (GPL3).

Drug screening and genome editing in human pancreatic cancer organoids identifies drug-gene interactions and candidates for off-label treatment.

Pancreatic cancer (PDAC) is a highly aggressive malignancy for which the identification of novel therapies is urgently needed. Here, we establish a human PDAC organoid biobank from 31 genetically distinct lines, covering a representative range of tumor subtypes, and demonstrate that these reflect the molecular and phenotypic heterogeneity of primary PDAC tissue. We use CRISPR-Cas9 genome editing and drug screening to characterize drug-gene interactions with ARID1A and BRCA2. We find that missense- but not frameshift mutations in the PDAC driver gene ARID1A are associated with increased sensitivity to the kinase inhibitors dasatinib (p < 0.0001) and VE-821 (p < 0.0001). We conduct an automated drug-repurposing screen with 1,172 FDA-approved compounds, identifying 26 compounds that effectively kill PDAC organoids, including 19 chemotherapy drugs currently approved for other cancer types. We validate the activity of these compounds in vitro and in vivo. The in vivo validated hits include emetine and ouabain, compounds which are approved for non-cancer indications and which perturb the ability of PDAC organoids to respond to hypoxia. Our study provides proof-of-concept for advancing precision oncology and identifying candidates for drug repurposing via genome editing and drug screening in tumor organoid biobanks.

SwissMTB: establishing comprehensive molecular cancer diagnostics in Swiss clinics.

BACKGROUND: Molecular precision oncology is an emerging practice to improve cancer therapy by decreasing the risk of choosing treatments that lack efficacy or cause adverse events. However, the challenges of integrating molecular profiling into routine clinical care are manifold. From a computational perspective these include the importance of a short analysis turnaround time, the interpretation of complex drug-gene and gene-gene interactions, and the necessity of standardized high-quality workflows. In addition, difficulties faced when integrating molecular diagnostics into clinical practice are ethical concerns, legal requirements, and limited availability of treatment options beyond standard of care as well as the overall lack of awareness of their existence. METHODS: To the best of our knowledge, we are the first group in Switzerland that established a workflow for personalized diagnostics based on comprehensive high-throughput sequencing of tumors at the clinic. Our workflow, named SwissMTB (Swiss Molecular Tumor Board), links genetic tumor alterations and gene expression to therapeutic options and clinical trial opportunities. The resulting treatment recommendations are summarized in a clinical report and discussed in a molecular tumor board at the clinic to support therapy decisions. RESULTS: Here we present results from an observational pilot study including 22 late-stage cancer patients. In this study we were able to identify actionable variants and corresponding therapies for 19 patients. Half of the patients were analyzed retrospectively. In two patients we identified resistance-associated variants explaining lack of therapy response. For five out of eleven patients analyzed before treatment the SwissMTB diagnostic influenced treatment decision. CONCLUSIONS: SwissMTB enables the analysis and clinical interpretation of large numbers of potentially actionable molecular targets. Thus, our workflow paves the way towards a more frequent use of comprehensive molecular diagnostics in Swiss hospitals.