Identification and characterization of chemotherapy resistant high-risk neuroblastoma persister cells.

Relapse rates in high-risk neuroblastoma remain exceedingly high. The malignant cells that are responsible for relapse have not been identified, and mechanisms of therapy resistance remain poorly understood. Here, we used single nucleus RNA sequencing and bulk whole genome sequencing to identify and characterize the residual malignant persister cells that survive chemotherapy from a cohort of 20 matched diagnosis and definitive surgery tumor samples from patients treated with high-risk neuroblastoma induction chemotherapy. We show that persister cells share common mechanisms of chemotherapy escape including suppression of MYCN activity and activation of NF-κB signaling, the latter is further enhanced by cell-cell communication between the malignant cells and the tumor microenvironment. Overall, our work dissects the transcriptional landscape of cellular persistence in high-risk neuroblastoma and paves the way to the development of new therapeutic strategies to prevent disease relapse.

Mapping the cellular biogeography of human bone marrow niches using single-cell transcriptomics and proteomic imaging.

Non-hematopoietic cells are essential contributors to hematopoiesis. However, heterogeneity and spatial organization of these cells in human bone marrow remain largely uncharacterized. We used single-cell RNA sequencing (scRNA-seq) to profile 29,325 non-hematopoietic cells and discovered nine transcriptionally distinct subtypes. We simultaneously profiled 53,417 hematopoietic cells and predicted their interactions with non-hematopoietic subsets. We employed co-detection by indexing (CODEX) to spatially profile over 1.2 million cells. We integrated scRNA-seq and CODEX data to link predicted cellular signaling with spatial proximity. Our analysis revealed a hyperoxygenated arterio-endosteal neighborhood for early myelopoiesis, and an adipocytic localization for early hematopoietic stem and progenitor cells (HSPCs). We used our CODEX atlas to annotate new images and uncovered mesenchymal stromal cell (MSC) expansion and spatial neighborhoods co-enriched for leukemic blasts and MSCs in acute myeloid leukemia (AML) patient samples. This spatially resolved, multiomic atlas of human bone marrow provides a reference for investigation of cellular interactions that drive hematopoiesis.

Mapping the Cellular Biogeography of Human Bone Marrow Niches Using Single-Cell Transcriptomics and Proteomic Imaging.

The bone marrow is the organ responsible for blood production. Diverse non-hematopoietic cells contribute essentially to hematopoiesis. However, these cells and their spatial organization remain largely uncharacterized as they have been technically challenging to study in humans. Here, we used fresh femoral head samples and performed single-cell RNA sequencing (scRNA-Seq) to profile 29,325 enriched non-hematopoietic bone marrow cells and discover nine transcriptionally distinct subtypes. We next employed CO-detection by inDEXing (CODEX) multiplexed imaging of 18 individuals, including both healthy and acute myeloid leukemia (AML) samples, to spatially profile over one million single cells with a novel 53-antibody panel. We discovered a relatively hyperoxygenated arterio-endosteal niche for early myelopoiesis, and an adipocytic, but not endosteal or perivascular, niche for early hematopoietic stem and progenitor cells. We used our atlas to predict cell type labels in new bone marrow images and used these predictions to uncover mesenchymal stromal cell (MSC) expansion and leukemic blast/MSC-enriched spatial neighborhoods in AML patient samples. Our work represents the first comprehensive, spatially-resolved multiomic atlas of human bone marrow and will serve as a reference for future investigation of cellular interactions that drive hematopoiesis.

A longitudinal single-cell and spatial multiomic atlas of pediatric high-grade glioma.

Pediatric high-grade glioma (pHGG) is an incurable central nervous system malignancy that is a leading cause of pediatric cancer death. While pHGG shares many similarities to adult glioma, it is increasingly recognized as a molecularly distinct, yet highly heterogeneous disease. In this study, we longitudinally profiled a molecularly diverse cohort of 16 pHGG patients before and after standard therapy through single-nucleus RNA and ATAC sequencing, whole-genome sequencing, and CODEX spatial proteomics to capture the evolution of the tumor microenvironment during progression following treatment. We found that the canonical neoplastic cell phenotypes of adult glioblastoma are insufficient to capture the range of tumor cell states in a pediatric cohort and observed differential tumor-myeloid interactions between malignant cell states. We identified key transcriptional regulators of pHGG cell states and did not observe the marked proneural to mesenchymal shift characteristic of adult glioblastoma. We showed that essential neuromodulators and the interferon response are upregulated post-therapy along with an increase in non-neoplastic oligodendrocytes. Through in vitro pharmacological perturbation, we demonstrated novel malignant cell-intrinsic targets. This multiomic atlas of longitudinal pHGG captures the key features of therapy response that support distinction from its adult counterpart and suggests therapeutic strategies which are targeted to pediatric gliomas.

Identification and targeting of treatment resistant progenitor populations in T-cell Acute Lymphoblastic Leukemia.

Refractoriness to initial chemotherapy and relapse after remission are the main obstacles to cure in T-cell Acute Lymphoblastic Leukemia (T-ALL). Biomarker guided risk stratification and targeted therapy have the potential to improve outcomes in high-risk T-ALL; however, cellular and genetic factors contributing to treatment resistance remain unknown. Previous bulk genomic studies in T-ALL have implicated tumor heterogeneity as an unexplored mechanism for treatment failure. To link tumor subpopulations with clinical outcome, we created an atlas of healthy pediatric hematopoiesis and applied single-cell multiomic (CITE-seq/snATAC-seq) analysis to a cohort of 40 cases of T-ALL treated on the Children's Oncology Group AALL0434 clinical trial. The cohort was carefully selected to capture the immunophenotypic diversity of T-ALL, with early T-cell precursor (ETP) and Near/Non-ETP subtypes represented, as well as enriched with both relapsed and treatment refractory cases. Integrated analyses of T-ALL blasts and normal T-cell precursors identified a bone-marrow progenitor-like (BMP-like) leukemia sub-population associated with treatment failure and poor overall survival. The single-cell-derived molecular signature of BMP-like blasts predicted poor outcome across multiple subtypes of T-ALL within two independent patient cohorts using bulk RNA-sequencing data from over 1300 patients. We defined the mutational landscape of BMP-like T-ALL, finding that NOTCH1 mutations additively drive T-ALL blasts away from the BMP-like state. We transcriptionally matched BMP-like blasts to early thymic seeding progenitors that have low NR3C1 expression and high stem cell gene expression, corresponding to a corticosteroid and conventional cytotoxic resistant phenotype we observed in ex vivo drug screening. To identify novel targets for BMP-like blasts, we performed in silico and in vitro drug screening against the BMP-like signature and prioritized BMP-like overexpressed cell-surface (CD44, ITGA4, LGALS1) and intracellular proteins (BCL-2, MCL-1, BTK, NF-κB) as candidates for precision targeted therapy. We established patient derived xenograft models of BMP-high and BMP-low leukemias, which revealed vulnerability of BMP-like blasts to apoptosis-inducing agents, TEC-kinase inhibitors, and proteasome inhibitors. Our study establishes the first multi-omic signatures for rapid risk-stratification and targeted treatment of high-risk T-ALL.

Isolation of Epithelial and Stromal Cells from Colon Tissues in Homeostasis and Under Inflammatory Conditions.

Inflammation of the gastrointestinal tract is a prevalent pathology in diseases such as inflammatory bowel disease (IBD). Currently, there are no therapies to prevent IBD, and available therapies to treat IBD are often sub-optimal. Thus, an unmet need exists to better understand the molecular mechanisms underlying intestinal tissue responses to damage and regeneration. The recent development of single-cell RNA (sc-RNA) sequencing-based techniques offers a unique opportunity to shed light on novel signaling pathways and cellular states that govern tissue adaptation or maladaptation across a broad spectrum of diseases. These approaches require the isolation of high-quality cells from tissues for downstream transcriptomic analyses. In the context of intestinal biology, there is a lack of protocols that ensure the isolation of epithelial and non-epithelial compartments simultaneously with high-quality yield. Here, we report two protocols for the isolation of epithelial and stromal cells from mouse and human colon tissues under inflammatory conditions. Specifically, we tested the feasibility of the protocols in a mouse model of dextran sodium sulfate (DSS)-induced colitis and in human biopsies from Crohn's patients. We performed sc-RNA sequencing analysis and demonstrated that the protocol preserves most of the epithelial and stromal cell types found in the colon. Moreover, the protocol is suitable for immunofluorescence staining of surface markers for epithelial, stromal, and immune cell lineages for flow cytometry analyses. This optimized protocol will provide a new resource for scientists to study complex tissues such as the colon in the context of tissue damage and regeneration. Key features • This protocol allows the isolation of epithelial and stromal cells from colon tissues. • The protocol has been optimized for tissues under inflammatory conditions with compromised cell viability. • This protocol is suitable for experimental mouse models of colon inflammation and human biopsies.

PNC-27, a Chimeric p53-Penetratin Peptide Binds to HDM-2 in a p53 Peptide-like Structure, Induces Selective Membrane-Pore Formation and Leads to Cancer Cell Lysis.

PNC-27, a 32-residue peptide that contains an HDM-2 binding domain and a cell-penetrating peptide (CPP) leader sequence kills cancer, but not normal, cells by binding to HDM-2 associated with the plasma membrane and induces the formation of pores causing tumor cell lysis and necrosis. Conformational energy calculations on the structure of PNC-27 bound to HDM-2 suggest that 1:1 complexes form between PNC-27 and HDM-2 with the leader sequence pointing away from the complex. Immuno-scanning electron microscopy was carried out with cancer cells treated with PNC-27 and decorated with an anti-PNC-27 antibody coupled to 6 nm gold particles and an anti-HDM-2 antibody linked to 15 nm gold particles. We found multiple 6 nm- and 15 nm-labeled gold particles in approximately 1:1 ratios in layered ring-shaped structures in the pores near the cell surface suggesting that these complexes are important to the pore structure. No pores formed in the control, PNC-27-treated untransformed fibroblasts. Based on the theoretical and immuno-EM studies, we propose that the pores are lined by PNC-27 bound to HDM-2 at the membrane surface with the PNC-27 leader sequence lining the pores or by PNC-27 bound to HDM-2.

Molecular Targeting of H/MDM-2 Oncoprotein in Human Colon Cancer Cells and Stem-like Colonic Epithelial-derived Progenitor Cells.

BACKGROUND/AIM: We have tested whether the anticancer peptide, PNC-27, that kills cancer cells but not normal cells by binding to cancer cell membrane HDM-2 forming pores, kills CD44+ colon cancer stem cells. MATERIALS AND METHODS: Flow cytometry determined the CD44 and HDM-2 expression on six-colon cancer cell lines and one normal cell line (CCD-18Co). MTT, LDH release, annexin V binding and caspase 3 assays were used to assess PNC-27-induced cell death. Bioluminescence imaging measured PNC-27 effects on in vivo tumor growth. RESULTS: High percentages of cells in all six tumor lines expressed CD44. PNC-27 co-localized with membrane HDM-2 only in the cancer cells and caused total cell death (tumor cell necrosis, high LDH release, negative annexin V and caspase 3). In vivo, PNC-27 caused necrosis of tumor nodules but not of normal tissue. CONCLUSION: PNC-27 selectively kills colon cancer stem cells by binding of this peptide to membrane H/MDM-2.

Anti-Cancer Tumor Cell Necrosis of Epithelial Ovarian Cancer Cell Lines Depends on High Expression of HDM-2 Protein in Their Membranes.

OBJECTIVE: Patients with epithelial ovarian cancers experience the highest fatality rates among all gynecological malignancies which require development of novel treatment strategies. Tumor cell necrosis was previously reported in a number of cancer cell lines following treatment with a p53-derived anti-cancer peptide called PNC-27. This peptide induces necrosis by transmembrane pore formation with HDM-2 protein that is expressed in the cancer cell membrane. We aimed to extend these studies further by investigating expression of membrane HDM-2 protein in ovarian cancer as it relates to susceptibility to PNC-27. PROCEDURES: Herein, we measured HDM-2 membrane expression in two ovarian cancer cell lines (SKOV-3 and OVCAR-3) and a non-transformed control cell line (HUVEC) by flow cytometric and western blot analysis. Immunofluorescence was used to visualize colocalization of PNC-27 with membrane HDM-2. Treatment effects with PNC-27 and control peptide were assessed using a MTT cell proliferation assay while direct cytotoxicity was measured by lactate dehydrogenase (LDH) release and induction of apoptotic markers; annexin V and caspase-3. RESULTS: HDM-2 protein was highly expressed and frequently detected in the membranes of SKOV-3 and OVCAR-3 cells; a prominent 47.6 kDa HDM-2 plasma membrane isoform was present in both cell lines whereas 25, 29, and 30 kDa isoforms were preferentially expressed in OVCAR-3. Notably, PNC-27 colocalized with HDM-2 in the membranes of both cancer cell lines that resulted in rapid cellular necrosis. In contrast, no PNC-27 colocalization and cytotoxicity was observed with non-transformed HUVEC demonstrating minimal expression of membrane HDM-2. CONCLUSIONS: Our results suggest that HDM-2 is highly expressed in the membranes of these ovarian cancer cell lines and colocalizes with PNC-27. We therefore conclude that the association of PNC-27 with preferentially expressed membrane HDM-2 isoforms results in the proposed model for the formation of transmembrane pores and epithelial ovarian cancer tumor cell necrosis, as previously described in a number of solid tissue and hematologic malignancies.

Targeting Membrane HDM-2 by PNC-27 Induces Necrosis in Leukemia Cells But Not in Normal Hematopoietic Cells.

BACKGROUND/AIM: Anticancer peptide PNC-27 binds to HDM-2 protein on cancer cell membranes inducing the formation of cytotoxic transmembrane pores. Herein, we investigated HDM-2 membrane expression and the effect of PNC-27 treatment on human non-stem cell acute myelogenous leukemia cell lines: U937, acute monocytic leukemia; OCI-AML3, acute myelomonocytic leukemia and HL60, acute promyelocytic leukemia. MATERIALS AND METHODS: We measured cell surface membrane expression of HDM-2 using flow cytometry. Cell viability was assessed using MTT assay while direct cytotoxicity was measured by lactate dehydrogenase (LDH) release and induction of apoptotic markers annexin V and caspase-3. RESULTS: HDM-2 is expressed at high levels in membranes of U937, OCI-AML3 and HL-60 cells. PNC-27 can bind to membrane HDM-2 to induce cell necrosis and LDH release within 4 h. CONCLUSION: Targeting membrane HDM-2 can be a potential strategy to treat leukemia. PNC-27 targeting membrane HDM-2 demonstrated significant anti-leukemia activity in a variety of leukemic cell lines.

Slippery Nanoparticles as a Diffusion Platform for Mucin Producing Gastrointestinal Tumors.

BACKGROUND: Treatment failure in pseudomyxoma peritonei (PMP) is partly attributed to the ineffective delivery of therapeutics through dense mucinous tumor barriers. We modified the surface of Poly (lactic-co-glycolic acid)-b-polyethylene glycol (PLGA-PEG-NPs) with a low-density, second PEG layer (PLGA-TPEG-NPs-20) to reduce their binding affinity to proteins and improve diffusion through mucin. METHODS: Nanoprecipitation was used to fabricate PLGA-PEG-NPs. To construct the second PEG layer of PLGA-TPEG-NPs-20, PEG-Thiol was conjugated to PLGA-PEG-NPs composed of 80% methoxy PLGA-PEG and 20% of PLGA-PEG-Maleimide. DiD-labeled nanoparticles (NPs) were added to the inner well of a trans-well system containing cultured LS174T or human PMP tissue. Diffusion of NPs was measured via fluorescence signal in the bottom well. In an ex vivo rat model, small intestine was treated with DiD-labeled NPs. In an in vivo murine LS174T subcutaneous tumor model, Nu/Nu nude mice received supratumoral injections (subcutaneous injection above the tumor) of DiD-labeled NPs. Thirty minutes after injection, mice were sacrificed, and tumors were collected. All tissue was cryosectioned, mounted with DAPI-containing media, and inspected via confocal microscopy. RESULTS: Diffusion profiles of NPs through PMP and cultured LS174T cells were generated. PLGA-TPEG-NPs-20 diffused faster with ~ 100% penetration versus PLGA-PEG-NPs with ~ 40% penetration after 8 h. Increased diffusion of PLGA-TPEG-NPs-20 was further observed in ex vivo rat small intestine as evidenced by elevated luminal NP fluorescence signal on the luminal surface. Subcutaneous LS174T tumors treated with PLGA-TPEG-NPs-20 demonstrated greater diffusion of NPs, showing homogenous fluorescence signal throughout the tumor. CONCLUSIONS: PLGA-TPEG-NPs-20 can be an effective mucin penetrating drug delivery system.

Hepatocellular Carcinoma: Etiology and Current and Future Drugs.

Hepatocellular carcinoma (HCC) is swiftly increasing in prevalence globally with a high mortality rate. The progression of HCC in patients is induced with advanced fibrosis, mainly cirrhosis, and hepatitis. The absence of proper preventive or curative treatment methods encouraged extensive research against HCC to develop new therapeutic strategies. The Food and Drug Administration-approved Nexavar (sorafenib) is used in the treatment of patients with unresectable HCC. In 2017, Stivarga (regorafenib) and Opdivo (nivolumab) got approved for patients with HCC after being treated with sorafenib, and in 2018, Lenvima (lenvatinib) got approved for patients with unresectable HCC. But, owing to the rapid drug resistance development and toxicities, these treatment options are not completely satisfactory. Therefore, there is an urgent need for new systemic combination therapies that target different signaling mechanisms, thereby decreasing the prospect of cancer cells developing resistance to treatment. In this review, HCC etiology and new therapeutic strategies that include currently approved drugs and other potential candidates of HCC such as Milciclib, palbociclib, galunisertib, ipafricept, and ramucirumab are evaluated.

Erratum: Thadi A.; et al. Early Investigations and Recent Advances in Intraperitoneal Immunotherapy for Peritoneal Metastasis. Vaccines 2018, 6, 54.

The authors wish to make the following corrections to this paper [...].

Early Investigations and Recent Advances in Intraperitoneal Immunotherapy for Peritoneal Metastasis.

Peritoneal metastasis (PM) is an advanced stage malignancy largely refractory to modern therapy. Intraperitoneal (IP) immunotherapy offers a novel approach for the control of regional disease of the peritoneal cavity by breaking immune tolerance. These strategies include heightening T-cell response and vaccine induction of anti-cancer memory against tumor-associated antigens. Early investigations with chimeric antigen receptor T cells (CAR-T cells), vaccine-based therapies, dendritic cells (DCs) in combination with pro-inflammatory cytokines and natural killer cells (NKs), adoptive cell transfer, and immune checkpoint inhibitors represent significant advances in the treatment of PM. IP delivery of CAR-T cells has shown demonstrable suppression of tumors expressing carcinoembryonic antigen. This response was enhanced when IP injected CAR-T cells were combined with anti-PD-L1 or anti-Gr1. Similarly, CAR-T cells against folate receptor α expressing tumors improved T-cell tumor localization and survival when combined with CD137 co-stimulatory signaling. Moreover, IP immunotherapy with catumaxomab, a trifunctional antibody approved in Europe, targets epithelial cell adhesion molecule (EpCAM) and has shown considerable promise with control of malignant ascites. Herein, we discuss immunologic approaches under investigation for treatment of PM.

Oral treatment with plecanatide or dolcanatide attenuates visceral hypersensitivity via activation of guanylate cyclase-C in rat models.

AIM: To investigate the effects of plecanatide and dolcanatide on maintenance of paracellular permeability, integrity of tight junctions and on suppression of visceral hypersensitivity. METHODS: Transport of fluorescein isothiocyanate (FITC)-dextran was measured to assess permeability across cell monolayers and rat colon tissues. Effects of plecanatide and dolcanatide on the integrity of tight junctions in Caco-2 and T84 monolayers and on the expression and localization of occludin and zonula occludens-1 (ZO-1) were examined by immunofluorescence microscopy. Anti-nociceptive activity of these agonists was evaluated in trinitrobenzene sulfonic acid (TNBS)-induced inflammatory as well as in non-inflammatory partial restraint stress (PRS) rat models. Statistical significance between the treatment groups in the permeability studies were evaluated using unpaired t-tests. RESULTS: Treatment of T84 and Caco-2 monolayers with lipopolysaccharide (LPS) rapidly increased permeability, which was effectively suppressed when monolayers were also treated with plecanatide or dolcanatide. Similarly, when T84 and Caco-2 monolayers were treated with LPS, cell surface localization of tight junction proteins occludin and ZO-1 was severely disrupted. When cell monolayers were treated with LPS in the presence of plecanatide or dolcanatide, occludin and ZO-1 were localized at the cell surface of adjoining cells, similar to that observed for vehicle treated cells. Treatment of cell monolayers with plecanatide or dolcanatide without LPS did not alter permeability, integrity of tight junctions and cell surface localization of either of the tight junction proteins. In rat visceral hypersensitivity models, both agonists suppressed the TNBS-induced increase in abdominal contractions in response to colorectal distension without affecting the colonic wall elasticity, and both agonists also reduced colonic hypersensitivity in the PRS model. CONCLUSION: Our results suggest that activation of GC-C signaling might be involved in maintenance of barrier function, possibly through regulating normal localization of tight junction proteins. Consistent with these findings, plecanatide and dolcanatide showed potent anti-nociceptive activity in rat visceral hypersensitivity models. These results imply that activation of GC-C signaling may be an attractive therapeutic approach to treat functional constipation disorders and inflammatory gastrointestinal conditions.

Plecanatide-mediated activation of guanylate cyclase-C suppresses inflammation-induced colorectal carcinogenesis in Apc+/Min-FCCC mice.

AIM: To evaluate the effect of orally administered plecanatide on colorectal dysplasia in Apc+/Min-FCCC mice with dextran sodium sulfate (DSS)-induced inflammation. METHODS: Inflammation driven colorectal carcinogenesis was induced in Apc+/Min-FCCC mice by administering DSS in their drinking water. Mice were fed a diet supplemented with plecanatide (0-20 ppm) and its effect on the multiplicity of histopathologically confirmed polypoid, flat and indeterminate dysplasia was evaluated. Plecanatide-mediated activation of guanylate cyclase-C (GC-C) signaling was assessed in colon tissues by measuring cyclic guanosine monophosphate (cGMP) by ELISA, protein kinase G-II and vasodilator stimulated phosphoprotein by immunoblotting. Ki-67, c-myc and cyclin D1 were used as markers of proliferation. Cellular levels and localization of ß-catenin in colon tissues were assessed by immunoblotting and immunohistochemistry, respectively. Uroguanylin (UG) and GC-C transcript levels were measured by quantitative reverse transcription polymerase chain reaction (RT-PCR). A mouse cytokine array panel was used to detect cytokines in the supernatant of colon explant cultures. RESULTS: Oral treatment of Apc+/MinFCCC mice with plecanatide produced a statistically significant reduction in the formation of inflammation-driven polypoid, flat and indeterminate dysplasias. This anti-carcinogenic activity of plecanatide was accompanied by activation of cGMP/GC-C signaling mediated inhibition of Wnt/ß-catenin signaling and reduced proliferation. Plecanatide also decreased secretion of pro-inflammatory cytokines (IL-6, IL1 TNF), chemokines (MIP-1, IP-10) and growth factors (GCSF and GMCSF) from colon explants derived from mice with acute DSS-induced inflammation. The effect of plecanatide-mediated inhibition of inflammation/dysplasia on endogenous expression of UG and GC-C transcripts was measured in intestinal tissues. Although GC-C expression was not altered appreciably, a statistically significant increase in the level of UG transcripts was detected in the proximal small intestine and colon, potentially due to a reduction in intestinal inflammation and/or neoplasia. Taken together, these results suggest that reductions in endogenous UG, accompanied by dysregulation in GC-C signaling, may be an early event in inflammation-promoted colorectal neoplasia; an event that can potentially be ameliorated by prophylactic intervention with plecanatide. CONCLUSION: This study provides the first evidence that orally administered plecanatide reduces the multiplicity of inflammation-driven colonic dysplasia in mice, demonstrating the utility for developing GC-C agonists as chemopreventive agents.