825 High-dimensional image cytometry reveals spatially organized tumor-immune microenvironment in hepatocellular carcinoma

BackgroundStructured and spatial-nuanced interactions between components in tumor microenvironment (TME) regulates the efficacy of anti-tumor regimens. Insights into this orchestrated behavior in therapeutic responders and non-responders will facilitate immunotherapies. High-multiplex imaging and spatial statistics enable deep profiling of TMEs by simultaneous arraying cell phenotypes and locations. In this study, we quantified the landscape of TMEs from neoadjuvant cabozantinib and nivolumab administered locally advanced hepatocellular carcinoma (HCC) biospecimen.Methods14 patients with HCC were treated with the combination of cabozantinib and nivolumab through the Johns Hopkins Sidney Kimmel Comprehensive Cancer Center. Among them, 12 patients (5 responders + 7 non-responders) underwent successful margin negative resection and are subjects to tissue microarray (TMAs) construction containing 37 representative tumor region cores. Using the TMAs, we performed imaging mass cytometry (IMC) with a panel of 27-cell lineage and functional markers (figure 1). All multiplexed images were then segmented to generate a single-cell dataset that enables (1) tumor-immune compartment analysis and (2) cell community analysis based on graph-embedding technology. Results from these hierarchies are merged to response-associated biological process patterns.ResultsImage processing on 37 multiplexed images discriminated 59,453 cells and then clustered into 17 cell types. Multi-level spatial quantification revealed distinct TME arrangements across cores from responders (R) and non-responders (NR): compartment analysis showed that at immune-tumor boundaries from NR, PD-L1 level on tumor cells is significantly higher than remote regions; however, Granzyme B level is lower (figure 2B). We also identified the proximity of CD8+ T cells to a subset of macrophages – Arginase 1hi CD163- macrophages (hazard macrophage) and CD4+ T cells, is a prognostic biomarker to neoadjuvant therapy (figure 3A and 3B). In-depth cell community analysis extracted cell-cell interaction networks based on spatial proximity. Next, hierarchical clustering grouped all networks with similar components (cell types) into 8 community categories (CC). Using graph-embedding and correlation test, we observed that in NR, macrophage-enriched CC (MCC) and lymphocytes-enriched CC (LCC) are strongly communicating with tumor CC; whereas in R, such communications were weakened by the engagement between MCC and LCC (figure 3C).ConclusionsIn conclusion, we employed an unbiased, quantitative spatial analysis to determine how tumor and immune components interact in responding and nonresponding HCC tumors. Based on our results, four immune-regulating factors are derived and summarized as a communication landscape (figure 4). The proposed framework represents a novel application of multiplexed imaging in translational medicine and has potential in initialization and validation of computational immuno-oncology models.AcknowledgementsThe authors acknowledge financial support from Bristol-Myers Squibb, Exelixis, the National Cancer Institute Specialized Program of Research Excellence (SPORE) in Gastrointestinal Cancers (P50 CA062924), the Passano Foundation, the National Institutes of Health (Grant No. U01CA212007 and R01CA138264) and Emerson Collective Cancer Research Fund (640183).Ethics ApprovalThe studies involving human participants were reviewed and approved by Institutional Review Board of the Johns Hopkins Medical Institutions.ConsentWritten informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.Abstract 825 Figure 1A panel of 27 markers was used to stain the hepatocellular carcinoma tumor region cores and processed using IMC. The marker names and descriptions are includedAbstract 825 Figure 2(A) Color overlays of lineage proteins covering Pan-Keratin and CD45 (rainbow) and functional markers covering PD-L1 and Granzyme B (white) in whole tissue core and subregions. (B) and (C) Protein expression analytical strategy. For compartmentalized cores, functional marker expressions on target cells were examined adjacent and remote to tumor-immune border and truncated to treatment response criteria for comparisonsAbstract 825 Figure 3(A) Diagram of CD8+ T cell RiskScore. Denote each CD8+ T cell to its nearest hazard macrophages as d1 and to its nearest CD4+ T cell as d2, thus the RiskScore is formally computed by taking the proportion of d2 to the combined distance of d1 and d2. (B) RiskScore on per-cell basis for responders and non-responders. (C) Cell community communication maps in tumor microenvironment associated with responders and non-respondersAbstract 825 Figure 4The synergistic anti-tumor immunity of macrophages and lymphocytes favors cabozantinib and nivolumab; immune function regulators (i.e., GranB and PD-L1) were upregulated throughout the immune compartment in non-responders; close proximity to hazard macrophages and distance away from CD4+ T cells associate with poorer effector function of CD8+ T cells

WDR31 is a novel ciliopathy protein displaying functional redundancy with GTPase-activating proteins ELMOD and RP2 in recruiting BBSome to cilium

The term “ciliopathy” refers to a group of over 35 rare disorders characterized by defective cilia and many overlapping clinical features, such as hydrocephalus, cerebellar vermis hypoplasia, polydactyly, and retinopathy. Even though many genes have been implicated in ciliopathies, the genetic pathogenesis in certain cases remains still undisclosed. Here, we identified a homozygous truncating variant in WDR31 in a patient with a typical ciliopathy phenotype encompassing congenital hydrocephalus, polydactyly, and renal agenesis. WDR31 is an evolutionarily conserved protein that localizes to the cilium and cilia-related compartment. Analysis from zebrafish supports the role of WDR31 in regulating the cilia morphology. The CRISPR/Cas9 knock-in (p.Arg261del) C. elegans model of the patient variant (p.Arg268*) reproduced several cilia-related defects observed in wdr-31 null mutants. Mechanistic analysis from C. elegans revealed that WDR-31 functions redundantly with ELDM-1 (ELMOD protein) and RPI-2 (RP2) to regulate the IFT trafficking through controlling the cilia entry of the BBSome. This work revealed WDR31 as a new ciliopathy protein that regulates IFT and BBSome trafficking.

WDR31 is a novel ciliopathy protein displaying functional redundancy with GTPase-activating proteins ELMOD and RP2 in recruiting BBSome to cilium

The term ciliopathy refers to a group of over 35 rare disorders characterized by defective cilia and many overlapping clinical features, such as hydrocephalus, cerebellar vermis hypoplasia, polydactyly, and retinopathy. Even though many genes have been implicated in ciliopathies, the genetic pathogenesis in certain cases remains still undisclosed. Here, we identified a homozygous truncating variant in WDR31 in a patient with a typical ciliopathy phenotype encompassing congenital hydrocephalus, polydactyly and renal agenesis. WDR31 is an evolutionarily conserved protein that localizes to the cilium and cilia-related compartment. Analysis from zebrafish supports the role of WDR31 in regulating the cilia morphology. The CRISPR/Cas9 knock-in (p.Arg261del) C. elegans model of the patient variant (p.Arg268*) reproduced several cilia-related defects observed in wdr-31 null mutants. Mechanistic analysis from C. elegans revealed that WDR-31 functions redundantly with ELDM-1 (ELMOD protein) and RPI-2 (RP2) to regulate the IFT trafficking through controlling the cilia entry of the BBSome. This work revealed WDR31 as a new ciliopathy protein that regulates IFT and BBSome trafficking.

The metastasis suppressor protein NM23-H1 modulates the PI3K-AKT axis through interaction with the p110α catalytic subunit

The PI3K pathway is one of the most deregulated pathways in cancer, which is predominantly due to gain of function mutations or altered expression of the PI3KCA gene. This is codified by what is seen for the class I PI3K catalytic subunit p110α, a common feature of many cancers. The metastasis suppressor protein NM23-H1 (NME1), whose ability to suppress the metastasis activities of different tumors has been widely described and was previously reported to alter phosphatidylinositol signaling. Here, we show interaction of NM23-H1 with the p110α subunit and the functional consequence of this interaction. This interaction is predominantly localized at the plasma membrane with some signals seen in the cytoplasmic compartment. Analysis of NM23-H1 levels showed a negative correlation between NM23-H1 expression and Akt phosphorylation, the key marker of PI3K pathway activation. Investigating the functional consequence of this interaction using cell motility and clonogenicity assays showed that expression of NM23-H1 reversed the enhanced migration, invasion, adhesion, and filopodia structure formation in cells expressing the p110α catalytic subunit. A similar trend was seen in anchorage-independent assays. Notably, differential analyses using NM23-H1 mutants which lacked the enzymatic and metastasis suppressor activity, showed no detectable interaction between p110α and the NM23-H1 mutant proteins P96S, H118F, and S120G, as well as no dysregulation of the PI3K-AKT axis.

dcHiC: differential compartment analysis of Hi-C datasets

Compartmental organization plays a role in important biological processes. However, its comparative analysis has been mainly limited to pairwise comparisons of contact maps with focus on finding compartment flips (e.g., A-to-B). Here, we introduce dcHiC, which utilizes Multiple Factor Analysis and a multivariate distance measure to systematically identify all compartmentalization differences among multiple contact maps. Evaluating dcHiC on three different collections of Hi-C data, we show its effectiveness and sensitivity in detecting biologically meaningful differences associated with cellular identity, gene expression, and lamin association. By providing a multivariate formulation, dcHiC immediately expands compartment analysis to new modalities in comparative genomics.

Cellular compartment analysis of temporal activity by fluorescent in situ hybridization (catFISH) in the transcardially perfused rat brain

Cellular compartment analysis of temporal activity by fluorescent in situ hybridization (catFISH) allows high spatiotemporal resolution mapping of immediate early genes in the brain in response to internal/external stimuli. One caveat of this technique and indeed other methods of in situ hybridization is the necessity of flash-freezing the brain prior to staining. Often however, the mammalian brain is transcardially perfused to use the brain tissue for immunohistochemistry, the most widely-used technique to study gene expression. The present study illustrates how the original catFISH protocol can be modified for use in adult rats that have been transcardially perfused with 4% paraformaldehyde. c-Fos activity induced by either an auditory tone or status epilepticus was visualized using the catFISH procedure. Analysis of the rat prefrontal cortex, hippocampus and amygdala shows that a clear distinction can be made between the compartmental distribution of c-Fos mRNA in the nuclei and cytoplasmic regions. Furthermore, the qualitative proportion of c-Fos compartmentalization is similar to previous reports of c-Fos expression pattern in rodents navigating novel environments. c-Fos catFISH on perfused rodent brains is an valuable addition to the traditional histological methods using fluorescently labeled riboprobes, and opens several avenues for future investigations.