Trussoni, Christy E.

Abstract Human genomic studies link reduced CUB domain-containing protein 1 (CDCP1) expression with myocardial recovery in heart failure. While CDCP1 regulates cardiac fibroblast proliferation in vitro, it’s in vivo role in cardiac fibrosis remains unclear. Using a Cdcp1 -knockout (KO) angiotensin II/phenylephrine mouse model, we show that Cdcp1 deletion reduces echocardiographic left ventricular mass, histologic cardiac fibrosis, and pro-fibrotic gene expression, along with decreased fibroblast activation and inflammatory markers. Spatial transcriptomics identified a pressure overload–expanded fibroblast subpopulation enriched for growth factor and TGF-β signaling (FB5), which was markedly attenuated in Cdcp1 -KO hearts, alongside reduction of a pro-inflammatory cardiomyocyte subtype (CM4). Complementary studies in human ventricular fibroblasts demonstrate that CDCP1 knockdown reduced extracellular matrix gene expression and collagen I deposition. These findings establish CDCP1 as a regulator of cardiac fibrotic remodeling in vivo and open avenues for its further investigation as a potential therapeutic target.

Abstract The importance of bone marrow (BM) microenvironment and its immune interactions within and their role in chronic myelomonocytic leukemia (CMML) disease progression have not been well studied. Our previous work in CMML identified type 1 helper T cell (Th1) and CD4 terminal effector cell suppression in peripheral blood from untreated patients, in comparison to age-matched normal controls (Mangaonkar A et al. Blood Adv 2020). Furthermore, clonal dendritic cell (DC) aggregates in CMML are present in approximately 20-30% patients at diagnosis, display significant cell heterogeneity (plasmacytoid DCs + myeloid DCs and other myeloid/immune cells), express indoleamine 2,3-dioxygenase-1 (IDO), associate with T regulatory cell (Treg) expansion, and transformation to AML [blast transformation or BT, Lucas N et al. Leukemia 2019, Mangaonkar A et al. Blood Adv 2020, and Mangaonkar A et al. Blood 2023 (abstract)]. The purpose of this study was to define the myeloid-immune cell interactions within the BM microenvironment critical for CMML progression to AML. To phenotypically identify cell types and their interactions within the CMML BM, we used single-cell type spatial omics through two distinct modalities on archival formalin-fixed paraffin-embedded (FFPE) BM biopsy specimens. Ten serial CMML samples at chronic phase [CP, n=5, median BM blast% 5 (2-15)] and at BT [n=5, median BM blast% 60 (25-74)] were used for both techniques. Spatial transcriptomics was conducted through GeoMx Digital Spatial Profiler (DSP) platform that uses UV-photocleavable, spatial-barcoded oligo probes to profile the whole transcriptome in defined regions of interest (ROI). CD123 (marker for DC-myeloid aggregates) immunofluorescence antibody was used to identify and segment ROI. 16-color flow cytometry was done to validate cell-specific expression. Spatial cellular phenotyping of immune cells on the same samples (serial cuts) was done through multiplex imaging by co-detection by indexing (CODEX) using 27 markers: CD31, Ki67, CD11b, CD20, CD44, Ecad, CD123, CD107a, CD8, CD11c, CD45RO, FoxP3, CD14, PDL1, CD56, CD68, CD163, PD-1, CD38, HLA-DR, CD4, IDO, CD45, PanCK, Na/K/ATPase, CD3e, and DAPI. CD8T+ cell anergy was demonstrated through CFSE cell proliferation assay. Benjamini-Hochberg correction was used to adjust for multiple hypotheses. After filtering poor quality ROIs [ROIs with total read count < 1000 were removed, ROIs with trim, stitch, alignment < 80% or saturation < 50% were removed], 3rd-quartile normalization [BiocGenerics (v. 0.48.1)] and batch correction [limma (v. 3.58.1)], we compared transcriptomics signatures between CD123+ and CD123- ROIs in all samples. Results showed that LIPA, SLC4A1, HBA2, TIMD4 and HMOX1 werethe top five genes upregulated in CD123+ areas. Among all gene transcripts, TIMD4 expression increased the most (Log2FC=0.6, P-value=8.79E-10, FC=1.52, FDR=1.68E-07) when CD123+ ROIs were compared between CMML-CP and CMML-BT serial samples, suggesting a biological relevance in disease progression. Previous work in metastatic peritoneal cancer has established that TIM-4 expressing macrophages suppress Th1/Th2 ratios and impair CD8+ T cell function (Chow A et al. Cancer Cell 2021). To confirm expression at the protein level, we used a multicolor flow cytometry panel and confirmed that TIM4 is expressed on macrophages (Lineage-negative, HLA-DR+, CD68+ cells) in independent CMML BM aspirate samples (n=3). Next, we conducted spatial phenotyping using CODEX on serial cuts from the same serial samples used for spatial transcriptomics. Anatomical co-localization of macrophages with leukemic cells, monocytes and T cells was visually inspected and confirmed. Cell-interaction analysis (after Benjamini-Hochberg correction) indicated that macrophages-monocyte, macrophage-leukemic cells, and macrophage-memory CD4+T cells were significantly (P<0.05) increased in CMML-BT versus CMML-CP. CD8T+ cell anergy was confirmed through a CFSE cell proliferation assay in untreated CMML PBMC samples. In conclusion, our data suggests that TIM4-expressing macrophages and their interactions with monocytes/monocyte precursors and immune cells within a DC aggregate create a protective leukemic stem cell niche and T cell tolerance, promoting blast transformation in CMML.

Abstract Purpose: Digital proteomic profiling was performed to identify spatial context in relationship to patient response and survival after anti–PD-1 therapy in metastatic colorectal cancer. Experimental Design: Primary colorectal cancers with deficient mismatch repair from patients treated with anti–PD-1 antibodies were analyzed (N = 30) using digital spatial profiling (GeoMx nCounter). At the invasive margin, 71 proteins were profiled in 10 regions of interest/slide that were segmented into 3 compartments labeled with pan-cytokeratin (epithelia), CD45 (stromal cells), and SYTO13 (nuclei). In an independent cohort (n = 13), digital spatial profiling data and single-cell transcriptomic data were analyzed. Differential protein abundance, after Benjamini–Hochberg correction, was examined by clinical response and progression-free survival (PFS) using multivariable Cox regression. Results: Protein abundance varied significantly between epithelial and stromal compartments. Nonresponders to anti–PD-1 showed higher fibronectin and smooth muscle actin abundance in the epithelial compartment that was associated with significantly shorter PFS (adjusted HR: 6.49 and 4.52, respectively; P < 0.05). In CD45+ stroma, increased expression of proteins related to T cells (CD3 and CD4), NK cells (CD56), antigen presentation (CD40), immune activation (CD27, ICOS), and apoptosis (GZMA) were found in responders (vs nonresponders) to anti–PD-1; each marker was significantly associated with longer patient PFS (0.02 < adjusted HR < 0.17; P < 0.05). In a separate cohort, consistent results by compartment were found for fibronectin and CD56. Gene expression data revealed that fibronectin and smooth muscle actin were primarily derived from cancer-associated fibroblasts. Conclusions: Spatially resolved protein profiles within microenvironments of deficient mismatch repair colorectal cancers can influence patient response and survival after anti–PD-1, highlighting their potential clinical significance.

3520 Background: In patients with metastatic colorectal cancer (CRC) and deficient DNA mismatch repair (dMMR), we previously found transcriptomic signatures reflecting stromal compartment composition and proliferative capacity that can predict the efficacy of immune checkpoint blockade (ICB). To identify proteomic markers of response and/or resistance to ICB, we performed spatially resolved proteomic analysis of the tumor microenvironment. Methods: Patients with surgically resected, primary dMMR metastatic CRCs received pembrolizumab (Mayo Clinic, N=30). In FFPE tissues, Digital Spatial Profiling of 71 proteins was performed using panels covering immune cells, immune activation, cell death, PI3/AKT signaling, MAPK signaling, and pan-tumor markers (GeoMx nCounter). In ten regions of interest/slide (~600 µm2) at the tumor invasive margin, protein expression was determined in four cellular compartments: epithelium (panCK+), immune cells (CD45+; leucocyte common antigen), stroma (panCK-), and nonimmune stroma (panCK-/CD45-). After normalization, protein abundance per compartment was analyzed by response (RECIST 1.1) [OmicVerse Python]. Differentially expressed proteins (binary) were examined by progression-free survival (PFS) using multivariable Cox regression with covariate adjustment (Lifelines Python). Results: Pre-treatment panels including 71 proteins were analyzed by treatment response and PFS after PD-1 inhibition. Proteins found to be significantly associated with response (p< 0.01) and PFS are described. Across all compartments, responders showed upregulation of NK cells and T cell subsets (CD3+, CD4+, CD8+), and ICOS; strongest association was found for CD4+: HRadj: 0.16; 95%CI: 0.08-0.31; p< 0.001. Nonresponders showed higher phospho-JNK (HRadj: 2.09; 95%CI: 1.28-3.42; p=0.003) and SMA. In the epithelial compartment, upregulation of the Treg marker CD25 and reduced caspase-9 were observed in responders. Among responders, the stromal compartment was highly enriched in HLA-DR, dendritic cells (CD11c), PD-L1, immune activation markers (CD40, CD44, CD80, CD27), CD95/Fas, and GZMB. In the stromal compartment, downregulation of BCL6 and phospho-GSK3B was observed in responders. The immune compartment of responders showed increased beta2-microglobulin and Ki-67, while the non-immune stromal compartment showed increase in the macrophage marker CD68. Importantly, proteins found to be upregulated in responders were significantly and independently associated with longer PFS whereas the opposite was true for nonresponders. Conclusions: In metastatic dMMR CRCs, spatially defined protein markers were significantly associated with anti-PD-1 response, and independently associated with patient PFS (strongest predictive utility for CD4 in immune stroma and phospho-JNK in epithelium).

Abstract Backgrounds and Aims Polycystic liver disease (PLD) is characterized by defective cholangiocyte cilia that regulate progressive growth of hepatic cysts. Because formation of primary cilia is influenced by autophagy through degradation of proteins involved in ciliogenesis, we hypothesized that ciliary defects in PLD cholangiocytes (PLDCs) originate from autophagy‐mediated depletion of ciliogenic proteins ADP‐ribosylation factor‐like protein 3 (ARL3) and ADP‐ribosylation factor‐like protein 13B (ARL13B) and ARL‐dependent mislocation of a ciliary‐localized bile acid receptor, Takeda G‐protein‐coupled receptor 5 (TGR5), the activation of which enhances hepatic cystogenesis (HCG). The aims here were to determine whether: (1) ciliogenesis is impaired in PLDC, is associated with increased autophagy, and involves autophagy‐mediated depletion of ARL3 and ARL13B; (2) depletion of ARL3 and ARL13B in PLDC cilia impacts ciliary localization of TGR5; and (3) pharmacological inhibition of autophagy re‐establishes cholangiocyte cilia and ciliary localization of ARL3, ARL3B, and TGR5 and reduces HCG. Approach and Results By using liver tissue from healthy persons and patients with PLD, in vitro and in vivo models of PLD, and in vitro models of ciliogenesis, we demonstrated that, in PLDCs: ciliogenesis is impaired; autophagy is enhanced; ARL3 and ARL13B are ubiquitinated by HDAC6, depleted in cilia, and present in autophagosomes; depletion of ARL3 and ARL13B impacts ciliary localization of TGR5; and pharmacological inhibition of autophagy with mefloquine and verteporfin re‐establishes cholangiocyte cilia and ciliary localization of ARL3, ARL13B, and TGR5 and reduces HCG. Conclusions The intersection between autophagy, defective cholangiocyte cilia, and enhanced HCG contributes to PLD progression and can be considered a target for therapeutic interventions.

AbstractBackground & AimsPrimary sclerosing cholangitis (PSC) is a chronic liver disease characterized by peribiliary inflammation and fibrosis. Cholangiocyte senescence is a prominent feature of PSC. Here, we hypothesize that extracellular vesicles (EVs) from senescent cholangiocytes influence the phenotype of target cells.MethodsEVs were isolated from normal human cholangiocytes (NHCs), cholangiocytes from PSC patients and NHCs experimentally induced to senescence. NHCs, malignant human cholangiocytes (MHCs) and monocytes were exposed to 108 EVs from each donor cell population and assessed for proliferation, MAPK activation and migration. Additionally, we isolated EVs from plasma of wild‐type and Mdr2−/− mice (a murine model of PSC), and assessed mouse monocyte activation.ResultsEVs exhibited the size and protein markers of exosomes. The number of EVs released from senescent human cholangiocytes was increased; similarly, the EVs in plasma from Mdr2−/− mice were increased. Additionally, EVs from senescent cholangiocytes were enriched in multiple growth factors, including EGF. NHCs exposed to EVs from senescent cholangiocytes showed increased NRAS and ERK1/2 activation. Moreover, EVs from senescent cholangiocytes promoted proliferation of NHCs and MHCs, findings that were blocked by erlotinib, an EGF receptor inhibitor. Furthermore, EVs from senescent cholangiocytes induced EGF‐dependent Interleukin 1‐beta and Tumour necrosis factor expression and migration of human monocytes; similarly, Mdr2−/− mouse plasma EVs induced activation of mouse monocytes.ConclusionsThe data continue to support the importance of cholangiocyte senescence in PSC pathogenesis, directly implicate EVs in cholangiocyte proliferation, malignant progression and immune cell activation and migration, and identify novel therapeutic approaches for PSC.

Cholangiocytes are the target of a group of chronic liver diseases termed the “cholangiopathies,” in which cholangiocytes react to exogenous and endogenous insults, leading to disease initiation and progression. In primary sclerosing cholangitis (PSC), the focus of this review, the cholangiocyte response to genetic or environmental insults can lead to a heterogeneous response; that is, a subpopulation acquires a ductular reactive and proliferative phenotype, while another subpopulation undergoes senescence and growth arrest. Both ductular reactive cholangiocytes and senescent cholangiocytes can modify the periductal microenvironment through their ability to secrete various cytokines, chemokines, and growth factors, initiating and perpetuating inflammatory and profibrotic responses. This review discusses the similarities and differences, the interrelationships, and the potential pathogenic roles of these reactive proliferative and senescent cholangiocyte subpopulations in PSC.

Cholangiocytes normally express primary cilia, a multisensory organelle that detects signals from the cellular environment. Cilia are significantly reduced in cholangiocarcinoma (CCA) by a mechanism involving overexpression of histone deacetylase 6 (HDAC6). Despite HDAC6 overexpression in CCA, we found no differences in its mRNA level, suggesting a posttranscriptional regulation, possibly involving microRNAs (miRNAs). Here, we describe that at least two HDAC6‐targeting miRNAs, miR‐433 and miR‐22, are down‐regulated in CCA both in vitro and in vivo. Experimental restoration of these miRNAs in CCA cells reduced HDAC6 expression, induced ciliary restoration, and decreased the malignant phenotype. Furthermore, in contrast to the mature forms, levels of precursor forms of these miRNAs were higher in CCA compared to normal cholangiocytes and accumulated in the nuclei, suggesting a defective nuclear export. We assessed the expression of Exportin‐5, the protein responsible for transporting miRNA precursors out of the nucleus, and found it to be reduced by 50% in CCA compared to normal cholangiocytes. Experimental overexpression of Exportin‐5 in CCA cells restored precursor and mature forms of these miRNAs to normal levels, inducing a decrease in the expression of HDAC6 and a decrease in the malignant phenotype. Conversely, short hairpin RNA (shRNA) depletion of Exportin‐5 in normal cholangiocytes resulted in increased nuclear retention of precursor miRNAs, decreased mature miRNAs, increased cell proliferation, and shorter cilia. Conclusion: These data suggest that down‐regulated Exportin‐5 impairs the nuclear export of miR‐433 and miR‐22 precursor forms, causing a decrease in levels of mature miR‐433 and miR‐22 forms, and leading to overexpression of HDAC6 and ciliary loss in CCA. (Hepatology 2018).