Hematogenous Cells Research Articles

A FACS-based novel isolation technique identifies heterogeneous CTCs in oral squamous cell carcinoma.

Isolating circulating tumour cells (CTCs) from the blood is challenging due to their low abundance and heterogeneity. Limitations of conventional CTC detection methods highlight the need for improved strategies to detect and isolate CTCs. Currently, the Food and Drug Administration (FDA)-approved CellSearch™ and other RUO techniques are not available in India. Therefore, we wanted to develop a flexible CTC detection/isolation technique that addresses the limitation(s) of currently available techniques and is suitable for various downstream applications. We developed a novel, efficient, user-friendly CTC isolation strategy combining density gradient centrifugation and immuno-magnetic hematogenous cell depletion with fluorescence-activated cell sorting (FACS)-based positive selection using multiple CTC-specific cell-surface markers. For FACS, a stringent gating strategy was optimised to exclude debris and doublets by side scatter/forward scatter (SSC/FSC) discriminator, remove dead cells by 4',6-diamidino-2-phenylindole (DAPI) staining, and eliminate non-specific fluorescence using a "dump" channel. APC-labelled anti-CD45mAB was used to gate remaining hematogenous cells, while multiple epithelial markers (EpCAM, EGFR, and Pan-Cytokeratin) and an epithelial-mesenchymal transition (EMT) marker (Vimentin) labelled with fluorescein isothiocyanate (FITC) were used to sort cancer cells. The technique was initially developed by spiking Cal 27 cancer cells into the blood of healthy donors and then validated in 95 biopsy-proven oral squamous cell carcinoma (OSCC) patients. CTCs isolated from patients were reconfirmed by Giemsa staining, immuno-staining, and whole transcriptome amplification (WTA), followed by qRT-PCR. In vitro culture and RNA sequencing (RNA-Seq) were also performed to confirm their suitability for various downstream applications. The mean detection efficiency for the Cal 27 tongue cancer cells spiked in the whole blood of healthy donors was 32.82% ± 12.71%. While ~75% of our patients (71/95) had detectable CTCs, the CTC positivity was independent of the TNM staging. The isolated potential cancer cells from OSCC patients were heterogeneous in size. They expressed different CTC-specific markers in various combinations as identified by qRT-PCR after WTA in different patients. Isolated CTCs were also found to be suitable for downstream applications like short-term CTC culture and RNA-Seq. We developed a sensitive, specific, flexible, and affordable CTC detection/isolation technique, which is scalable to larger patient cohorts, provides a snapshot of CTC heterogeneity, isolates live CTCs ready for downstream molecular analysis, and, most importantly, is suitable for developing countries.

A change of rhodocytin's suprastructure turns the agonist into an antagonist of tumor cell induced platelet aggregation.

Cancer cells circulating in the blood attach to platelets by direct cell-cell interactions via several receptor-counterreceptor contacts and indirectly by fibrin bridges which connect the two cell types by distinct integrin receptors. In the microenvironment of these tumor cell platelet aggregates (TCPAs), the tumor cells are shielded from the shear stress of the blood flow and from attack by the immune system. This supports hematogenous metastasis and tumor cell induced thrombosis. Platelet activation is triggered by binding of podoplanin on cancer cells to the platelet receptor Clec-2. Therefore, we hypothesize that targeting this initial step will prevent the entire cascade leading to the formation of TCPAs. Rhodocytin, a heterodimeric (αβ)2 C-type lectin from the Malayan pit viper Calloselasma rhodostoma, binds to Clec-2 and thereby induces TCPA formation. Remarkably, mutations in rhodocytin that disturbed formation of oligomers, blocked the podoplanin-Clec-2 axis and prevented platelet activation. Therefore, we used lysine reactive chemicals to modify rhodocytin isolated from the crude snake venom. Blue native gel electrophoresis and far western blotting showed a change of rhodocytin's suprastructure triggered by acetylation and PEGylation. Mass spectrometry analysis of altered lysines suggested that their modifications interfered with the formation of rhodocytin tetramers. When tested in assays for tumor cell induced platelet aggregation, we found that derivatization turned rhodocytin from an agonist into an antagonist. This observation indicates that Clec-2 is a valid target receptor molecule to curb TCPA formation and to prevent hematogenous metastasis and tumor cell induced thrombosis in cancer patients.

Hypothesis: hematogenous metastatic cancer cells of solid tumors may disguise themselves as memory macrophages for metastasis.

German pathologist Otto Aichel suggested, a century ago, that the cancer cell acquired its metastatic property from a leukocyte via cell-cell fusion. Since then, several revised versions of this theory have been proposed. Most of the proposals attribute the generation of the metastatic cancer cell to the fusion between a primary cancer cell and a macrophage. However, these theories have not addressed several issues, such as dormancy and stem cell-like self-renewal, of the metastatic cancer cell. On the other hand, recent studies have found that, like T- and B-/plasma cells, macrophages can also be categorized into naïve, effector, and memory/trained macrophages. As a memory/trained macrophage can enter dormancy/quiescence, be awakened from the dormancy/quiescence by acquainted primers, and re-populate via stem cell-like self-renewal, we, therefore, further specify that the macrophage fusing with the cancer cell and contributing to metastasis, belongs with the memory/trained macrophage, not other subtypes of macrophages. The current theory can explain many puzzling clinical features of cancer, including the paradoxal effects (recurrence vs. regression) of microbes on tumors, "spontaneous" and Coley's toxin-induced tumor regression, anticancer activities of β-blockers and anti-inflammatory/anti-immune/antibiotic drugs, oncotaxis, surgery- and trauma-promoted metastasis, and impact of microbiota on tumors. Potential therapeutic strategies, such as Coley's toxin-like preparations, are proposed. This is the last article of our trilogy on carcinogenesis theories.

ATROPHIC SCARS AS AN OBJECT OF INORGANIC GEL TREATMENT

Atrophic scars are a complex problem, covering a huge number of patients with striae gravidarum, acne, atrophic surgical scars, and striae due to hypercortisolism and other metabolic disorders. Taking into account patients’ social status (pregnant women, adolescents, people with chronic hormonal disorders), our goal was to systematize the literature data on the least invasive treatment modality – atrophic scar therapy with inorganic gels – to identify new chemical classes with potential anti-scar activity.
 When summarizing research data over the past 10 years, it was found that the effect of anti-scar therapy depends on the atrophic scar color – hyper- or hypopigmented, its type – wedge-shaped, scaphoid, or rectangular, its origin – post-acne, cicatricial alopecia, infectious scars, stretch marks, or post-traumatic atrophic. The stage of pathogenesis also plays a significant role: aseptic inflammation, infiltration of hematogenous differential cells, mast cell degranulation, release of bioactive substances and changes in the VEGF, TGF-β1, EGF, FGF, PDGF growth factors, migration of activated macrophages, damage to elastic and collagen fibers, perivascular lymphocytic infiltration, impaired hemorheology, chronic inflammatory process, increased number of senescent fibroblasts, decreased synthetic activity of atrophic scar cells, decreased cellularity and vascularization of the atrophic scar area. In case of high bioinertness of inorganic gels, the anti-scar effect is achieved due to the exfoliating effect, epidermal-mesenchymal interactions, and the impact of the mesoporous gel structure on scar hydration. In addition to the well-known silicone gel, sol-gels based on aluminum dioxide have similar properties, which makes it possible to consider sol-gel as an alternative to silicone gel.

Hepatic passaging of NRAS-mutant melanoma influences adhesive properties and metastatic pattern

BackgroundLiver metastasis is a poor prognostic factor for treatment of advanced cutaneous melanoma with either immunotherapy or targeted therapies. In this study we focused on NRAS mutated melanoma, a cohort with high unmet clinical need.MethodsWT31 melanoma was repeatedly passaged over the liver after intravenous injections five times generating the subline WT31_P5IV. The colonization of target organs, morphology, vascularization and the gene expression profiles of metastases were analyzed.ResultsAfter intravenous injection lung metastasis was significantly decreased and a trend towards increased liver metastasis was detected for WT31_P5IV as compared to parental WT31. Besides, the ratio of lung to liver metastases was significantly smaller. Histology of lung metastases revealed reduced proliferation of WT31_P5IV in relation to WT31 while both size and necrotic areas were unaltered. Liver metastases of both sublines showed no differences in vascularization, proliferation or necrosis. To identify tumor-intrinsic factors that altered the metastatic pattern of WT31_P5IV RNA sequencing was performed and revealed a differential regulation of pathways involved in cell adhesion. Ex vivo fluorescence imaging confirmed that initial tumor cell retention in the lungs was significantly reduced in WT31_P5IV in comparison to WT31.ConclusionThis study demonstrates that tumor-intrinsic properties influencing the metastatic pattern of NRAS mutated melanoma are strongly affected by hepatic passaging and the hematogenous route tumor cells take. It has implications for the clinical setting as such effects might also occur during metastatic spread or disease progression in melanoma patients.

Heliangin acts as a covalent ligand of RPS2 that disrupts pre-rRNA metabolic processes in NPM1-mutated acute myeloid leukemia.

Although NPM1 mutations are frequently found in acute myeloid leukemia patients, therapeutic strategies are scarce and unsuitable for those who cannot tolerate intensive chemotherapy. Here we demonstrated that heliangin, a natural sesquiterpene lactone, exerts favorable therapeutic responses in NPM1 mutant acute myeloid leukemia cells, with no apparent toxicity to normal hematogenous cells, by inhibiting their proliferation, inducing apoptosis, causing cell cycle arrest, and promoting differentiation. In-depth studies on its mode of action using quantitative thiol reactivity platform screening and subsequent molecular biology validation showed that the ribosomal protein S2 (RPS2) is the main target of heliangin in treating NPM1 mutant AML. Upon covalent binding to the C222 site of RPS2, the electrophilic moieties of heliangin disrupt pre-rRNA metabolic processes, leading to nucleolar stress, which in turn regulates the ribosomal proteins-MDM2-p53 pathway and stabilizes p53. Clinical data shows that the pre-rRNA metabolic pathway is dysregulated in acute myeloid leukemia patients with the NPM1 mutation, leading to a poor prognosis. We found that RPS2 plays a critical role in regulating this pathway and may be a novel treatment target. Our findings suggest a novel treatment strategy and lead compound for acute myeloid leukemia patients, especially those with NPM1 mutations.

Pro-metastatic and mesenchymal gene expression signatures characterize circulating tumor cells of neuroblastoma patients with bone marrow metastases and relapse.

Existing marker-based methods of minimal residual disease (MRD) determination in neuroblastoma do not effectively enrich for the circulating disease cell population. Given the relative size differential of neuroblastoma tumor cells over normal hematogenous cells, we hypothesized that cell size-based separation could enrich circulating tumor cells (CTCs) from blood samples and disseminated tumor cells (DTCs) from bone marrow aspirates (BMA) of neuroblastoma patients, and that their gene expression profiles could vary dynamically with various disease states over the course of treatment. Using a spiral microfluidic chip, peripheral blood of 17 neuroblastoma patients at 3 serial treatment timepoints (diagnosis, n=17; post-chemotherapy, n=11; and relapse, n=3), and bone marrow samples at diagnosis were enriched for large intact circulating cells. Profiling the resulting enriched samples with immunohistochemistry and mRNA expression of 1490 cancer-related genes via NanoString, 13 of 17 samples contained CTCs displaying cytologic atypia, TH and PHOX2B expression and/or upregulation of cancer-associated genes. Gene signatures reflecting pro-metastatic processes and the neuroblastoma mesenchymal super-enhancer state were consistently upregulated in 7 of 13 samples, 6 of which also had metastatic high-risk disease. Expression of 8 genes associated with PI3K and GCPR signaling were significantly upregulated in CTCs of patients with bone marrow metastases versus patients without. Correspondingly, in patients with marrow metastases, differentially-expressed gene signatures reflected upregulation of immune regulation in bone marrow DTCs versus paired CTCs samples. In patients who later developed disease relapse, 5 genes involved in immune cell regulation, JAK/STAT signaling and the neuroblastoma mesenchymal super-enhancer state (OLFML2B, STAT1, ARHGDIB, STAB1, TLR2) were upregulated in serial CTC samples over their disease course, despite urinary catecholamines and bone marrow aspirates not indicating the disease recurrences. In summary, using a label-free cell size-based separation method, we enriched and characterized intact circulating cells in peripheral blood indicative of neuroblastoma CTCs, as well as their DTC counterparts in the bone marrow. Expression profiles of pro-metastatic genes in CTCs correlated with the presence of bone marrow metastases at diagnosis, while longitudinal profiling identified persistently elevated expression of genes in CTCs that may serve as novel predictive markers of hematogenous MRD in neuroblastoma patients that subsequently relapse.

Immunomodulatory Strategies for Spinal Cord Injury.

Neuroinflammation is a key secondary event after spinal cord injury (SCI) and can increase barriers to regeneration, leading to various neurological disorders. Infiltrated hematogenous innate immune cells into the injured site are considered the main effector cells of the inflammatory responses after SCI. Glucocorticoids were the standard of care for spinal cord trauma for years due to their anti-inflammatory properties yet were also associated with unwanted side effects. While the administration of glucocorticoids is controversial, immunomodulatory strategies that limit inflammatory responses provide the potential therapeutic approaches to promote functional regeneration following SCI. Herein, we will discuss emerging therapeutic strategies to modulate inflammatory responses to enhance nerve recovery after spinal cord trauma.

Tumour cell-activated platelets modulate the immunological activity of CD4+, CD8+, and NK cells, which is efficiently antagonized by heparin

Platelets, key players in haemostasis, are progressively investigated with respect to their role in immunity and inflammation. Although the platelet support to haematogenous cancer cell metastasis has been the subject of multiple studies, their impact on anti-cancer immunity remains unaddressed. Here, we investigated the immunomodulatory potential of platelets upon their activation by MDA-MB-231 breast cancer cells in various in vitro approaches. We provide evidence that platelets as well as their tumour cell-induced releasates increased the ratio of regulatory T cells, shaping an immunosuppressive phenotype in isolated CD4+ cultures. The influence on CD8+ T cells was assessed by detecting the expression of activation markers CD25/CD69 and release of cytolytic and pro-inflammatory proteins. Notably, the platelet preparations differentially influenced CD8+ T cell activation, while platelets were found to inhibit the activation of CD8+ T cells, platelet releasates, in contrast, supported their activation. Furthermore, the NK cell cytolytic activity was attenuated by platelet releasates. Low molecular weight heparin (LMWH), the guideline-based anticoagulant for cancer-associated thrombotic events, is known to interfere with tumour cell-induced platelet activation. Thus, we aimed to investigate whether, unfractionated heparin, LMWH or novel synthetic heparin mimetics can also reverse the immunosuppressive platelet effects. The releasate-mediated alteration in immune cell activity was efficiently abrogated by heparin, while the synthetic heparin mimetics partly outperformed the commercial heparin derivatives. This is the first report on the effects of heparin on rebalancing immunosuppression in an oncological context emerging as a novel aspect in heparin anti-tumour activities.

Routing pathway of syngeneic donor hematopoietic stem cells after simple intra-amniotic delivery

BackgroundWe sought to determine the pathway through which syngeneic hematopoietic stem cells (HSCs) delivered into the amniotic fluid can reach the fetal circulation. MethodsLewis rat fetuses were divided in two groups based on the content of intra-amniotic injections performed on gestational day 17 (E17; term=E21–22): either a suspension of luciferase-labeled syngeneic HSCs (n = 137), or acellular luciferase (n = 44). Samples from placenta, chorion, amnion, amniotic fluid, umbilical cord, and 8 fetal sites were procured at 5 daily time points thereafter until term for analysis. ResultsWhen controlled by acellular luciferase, donor HSCs were identified in the amnion, chorion, placenta, and amniotic fluid of fetuses receiving cells at all time points (p = 0.033 to <0.001), peaking first at the amnion and subsequently at the chorion and placenta. Cells could be detected in the fetal liver as early as day 1, progressively expanding to all the other fetal sites over time, in parallel to their increased presence in the chorion and placenta. ConclusionsThe chronology of syngeneic donor hematopoietic stem cell trafficking after intra-amniotic injection is suggestive of controlled routing through the gestational membranes and placenta. Hematogenous donor cell routing is a constituent of transamniotic hematopoietic stem cell therapy, significantly expanding its potential applications.

Transcriptomic changes during stage progression of mycosis fungoides.

Mycosis fungoides (MF) is the most common cutaneous T-cell lymphoma, which in the early patch/plaque stages runs an indolent course. However, ~25% of patients with MF develop skin tumours, a hallmark of progression to the advanced stage, which is associated with high mortality. The mechanisms involved in stage progression are poorly elucidated. We sought to address the hypothesis of MF cell trafficking between skin lesions by comparing transcriptomic profiles of skin samples in different clinical stages of MF. We performed whole-transcriptome and whole-exome sequencing of malignant MF cells from skin biopsies obtained by laser-capture microdissection. We compared three types of MF lesions: early-stage plaques (ESP, n=12) as well as plaques and tumours from patients in late-stage disease [late-stage plaques (LSP, n=10) and tumours (TMR, n=15)]. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to determine pathway changes specific for different lesions which were linked to the recurrent somatic mutations overrepresented in MF tumours. The key upregulated pathways during stage progression were those related to cell proliferation and survival (MEK/ERK, Akt-mTOR), T helper cell (Th)2/Th9 signalling [interleukin (IL)4, STAT3, STAT5, STAT6], meiomitosis (CT45A1, CT45A3, STAG3, GTSF1, REC8) and DNA repair (PARP1, MYCN, OGG1). Principal coordinate clustering of the transcriptome revealed extensive gene expression differences between early (ESP) and advanced-stage lesions (LSP and TMR). LSP and TMR showed remarkable similarities at the level of the transcriptome, which we interpreted as evidence of cell percolation between lesions via haematogenous self-seeding. Stage progression in MF is associated with Th2/Th9 polarization of malignant cells, activation of proliferation, survival, as well as increased genomic instability. Global transcriptomic changes in multiple lesions may be caused by haematogenous cell percolation between discrete skin lesions.

Outcomes of Patients With Acute Myeloid Leukemia Who Relapse After 5 Years of Complete Remission.

Leukemia relapse 5 years after achieving first complete remission (CR1) is uncommon in patients with acute myeloid leukemia (AML). In this study, we evaluated the outcomes of AML patients with late relapse at our institution and reviewed the literature for these patients. The study cohort consisted of nine AML patients with late relapse. The median interval between CR1 and AML relapse was 6.1 years (range: 5.1–16.2 years). At relapse, the karyotype was different from the initial AML diagnosis in 50% of patients. At the time of AML relapse, seven patients received induction chemotherapy and two patients received hypomethylating agents with an overall CR rate of 66%. The median time to relapse after achieving second CR (CR2) was 16.5 months [95% confidence interval (CI): 9.4, NA]. The median overall survival after first relapse was 28.6 months (95% CI: 7.3, 3.4–66.5 months). Despite initial CR after reinduction therapy, relapse rates are still high, suggesting that alternative strategies for postremission therapies are warranted in CR2. These approaches include the use of allogeneic hematogenic cell transplantation and the use of newly approved AML agents as maintenance therapy in nontransplant eligible patients.

Outcomes with COVID-19 in hematopoietic stem cell transplant and cellular therapy patients.

7033 Background: The Coronavirus Disease 2019 (COVID-19) has caused over 25 million infections in the US with over 0.4 million deaths. Hematogenic stem cell transplant (HCT) or cellular therapy (CT) recipients have a high risk of mortality with COVID-19 due to profound immune dysregulation. We aimed to assess the outcomes with COVID-19 in HCT/CT recipients. Methods: A single-center prospective study was conducted, including all (n=40) adult HCT/CT patients who were diagnosed with COVID-19 at the University of Kansas from Apr 2020 to Jan 2021. Baseline and disease-related characteristics were ascertained from medical records. Data were analyzed using SPSS version 21 (SPSS Inc, Chicago, IL). Bivariate analyses, using chi-square and t-test, and logistic regression analyses were conducted. Results: The study included 40 COVID-19 patients (72.5% Oct 2020-Jan 2021), including allogeneic HCT (n=25), autologous HCT (n=13) and CAR-T CT (n=2) with median time since HCT/CT of 12.4 (1-201.9), 37.2 (0.4-118.7), and 3.8 (2.8-4.8) months. Seventy percent were Caucasians and 17.5 were Hispanics. Primary hematologic malignancy was myeloid (37.5%), lymphoid (35%) or plasma cell disorder (27.5%). Myeloablative conditioning was performed in 65% of patients. Donors were autologous (37.5%), matched sibling (17.5%), matched unrelated (22.5%) and haploidentical (22.5%). COVID-19 was mild (42.5%), moderate (42.5%) or severe (15%). Clinical findings included pneumonia (62.5%), hypoxia (25%) and ICU admission (17.5%) while therapies included remdesivir (47.5%), convalescent plasma (40%), dexamethasone (25%) and monoclonal antibodies (17.5%). Concurrent cancer treatment, other infections and active GVHD were reported in 25% (all myeloma), 20% and 32.5% of patients. After a median follow-up of 74 days (7-269), the mortality rate was 12.5% in all patients and 20% in allo-HCT patients. Significant predictors of COVID-19 severity included allogeneic HCT, concurrent immune suppression and elevated inflammatory markers. (Table). Conclusions: Hematopoietic stem cell transplant recipients have an increased risk of mortality with COVID-19. Our findings confirm the need for vaccination prioritization, close monitoring, and aggressive treatment in HCT/CT patients.[Table: see text]

Outcomes with COVID-19 in patients with hematopoietic stem cell transplant and cellular therapy: A systemic review and meta-analysis.

e18611 Background: Coronavirus Disease 2019 (COVID-19) was declared a pandemic on March 11, 2020. COVID-19 has caused over 100 million infections and over 2 million deaths globally. Patients who have received a hematogenic stem cell transplant or cellular therapy (HCT) have a high risk of mortality and morbidity with COVID-19 due to severe immune dysregulation. We conducted a systematic review and meta-analysis aimed to evaluate the outcomes of COVID-19 in HCT patients. Methods: A literature search following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines (PRISMA) guidelines was performed on 3 databases (PubMed, Cochrane, and Clinical trials.gov) from the date of inception to Jan 2021. MeSH terms included ‘hematological malignancies’, ‘hematopoietic stem cell transplantation’, ‘SARS-CoV-2’, and ‘COVID 19’. We screened 99 articles and 6 studies (4 retrospective studies, 2 prospective) were included after excluding review, duplicate, and non-relevant articles. Quality evaluation was done using the NIH quality assessment tool. The Inter-study heterogeneity among the studies was assessed using the Q statistic proposed by Cochrane and the I2 index introduced by Higgins and Thompson. Pooled analysis was done using the ‘metaXL’, and the random effects model was used to estimate the pooled prevalence with 95% CI. Results: Of 1619 patients in 6studies, 646 HCT patients were analyzed (Table ). The median age of patients was 63 years and 59% were males. Median days since HCT for autologous (auto) HCT and allogeneic (allo) HCT patients were 690 and 450 days respectively. The average follow-up duration after COVID-19 was 24 days. COVID-19 mortality in HCT patients was 20% (95%CI 0.17 to 0.23, I2=0). Roedl et al (n=6) reported a mortality of 83% and was excluded from the pooled analysis. The mortality rate was 19% (95% CI 0.15 to 0.24, I2=0%) in auto HCT patients and 21% (95% CI 0.17 to 0.25, I2=0%) in allo HCT patients. Conclusions: The HCT patients are at significant risk of increased mortality and morbidity due to COVID-19. There is a need to prioritize HCT patients for COVID-19 vaccination, close surveillance, and aggressive management.[Table: see text]

Multi-Arm PEG/Peptidomimetic Conjugate Inhibitors of DR6/APP Interaction Block Hematogenous Tumor Cell Extravasation.

The binding of amyloid precursor protein (APP) expressed on tumor cells to death receptor 6 (DR6) could initiate the necroptosis pathway, which leads to necroptotic cell death of vascular endothelial cells (ECs) and results in tumor cells (TCs) extravasation and metastasis. This study reports the first inhibitor of DR6/APP interaction as a novel class of anti‐hematogenous metastatic agent. By rationally utilizing three combined strategies including selection based on phage display library, d‐retro‐inverso modification, and multiple conjugation of screened peptidomimetic with 4‐arm PEG, the polymer–peptidomimetic conjugate PEG‐tAHP‐DRI (tetra‐(D‐retro‐inverso isomer of AHP‐12) substitued 4‐arm PEG5k) is obtained as the most promising agent with the strongest binding potency (K D = 51.12 × 10−9 m) and excellent pharmacokinetic properties. Importantly, PEG‐tAHP‐DRI provides efficient protection against TC‐induced ECs necroptosis both in vitro and in vivo. Moreover, this ligand exhibits prominent anti‐hematogenous metastatic activity in serval different metastatic mouse models (B16F10, 4T1, CT26, and spontaneous lung metastasis of 4T1 orthotopic tumor model) and displays no apparent detrimental effects in preliminary safety evaluation. Collectively, this study demonstrates the feasibility of exploiting DR6/APP interaction to regulate hematogenous tumor cells transendothelial migration and provides PEG‐tAHP‐DRI as a novel and promising inhibitor of DR6/APP interaction for developments of anti‐hematogenous metastatic therapies.

Anterior Approach vs Conventional Hepatectomy for Resection of Colorectal Liver Metastasis

Tumor relapse after partial hepatectomy for colorectal liver metastasis (CRLM) remains an unsolved issue. Intraoperative manipulation of the liver during conventional hepatectomy might enhance hematogenous tumor cell spread. The anterior approach is an alternative approach that may reduce intraoperative tumor cell dissemination. To determine the efficacy and safety of the anterior approach compared with conventional hepatectomy in patients undergoing resection for CRLM. This randomized clinical study evaluated the efficacy and safety of the anterior approach compared with conventional hepatectomy in adult patients with CRLM who were scheduled for hepatectomy from February 1, 2003, to March 31, 2012, at a tertiary-care hospital. A total of 80 patients with CRLM were randomized to the anterior approach and conventional hepatectomy groups in a 1:1 ratio. Bone marrow and blood samples were analyzed for disseminated tumor cells and circulating tumor cells (CTC) using cytokeratin 20 reverse transcriptase-polymerase chain reaction analysis. Data were analyzed from April 1 to December 1, 2018, using intention to treat. Anterior approach vs conventional hepatectomy. The primary end point was intraoperative CTC detection in central blood samples after liver resection. Secondary end points included postoperative morbidity, mortality, and long-term survival. Among the 80 patients included in the analysis (48 men [60%]; mean [SD] age, 61 [10] years), baseline characteristics, including preoperative CTC detection, were comparable between both groups. There was no statistically significant difference in intraoperative CTC detection between patients in the conventional hepatectomy (5 of 21 [24%]) and anterior approach (6 of 22 [27%]) groups (P = .54). Except for a longer operating time in the anterior approach group (mean [SD], 171 [53] vs 221 [53] minutes; P < .001), there were no significant differences in intraoperative and postoperative outcomes between both study groups. Although detection of CTC was associated with poor overall (median, 46 [95% CI, 40-52] vs 81 [95% CI, 54-107] months; P = .03) and disease-free (median, 40 [95% CI, 34-46] vs 60 [95% CI, 46-74] months; P = .04) survival, there was no significant difference in overall (median, 73 [95% CI, 42-104] vs 55 [95% CI, 35-75] months; P = .43) and disease-free (median, 48 [95% CI, 40-56] vs 40 [95% CI, 28-52] months; P = .88) survival between the conventional hepatectomy and anterior approach groups. Also, there was no significant difference in patterns of recurrence between both groups. This randomized clinical trial found that the anterior approach was not superior to conventional hepatectomy in reducing intraoperative tumor cell dissemination in patients undergoing resection of CRLM. isrctn.org Identifier: ISRCTN45066244.

Comparative Analysis of Blood and Bone Marrow for the Detection of Circulating and Disseminated Tumor Cells and Their Prognostic and Predictive Value in Esophageal Cancer Patients.

Hematogenic tumor cell spread is a key event in metastasis. However, the clinical significance of circulating tumor cells (CTC) in the blood and disseminated tumor cells (DTC) in bone marrow is still not fully understood. Here, the presence of DTC and CTC in esophageal cancer (EC) patients and its correlation with clinical parameters was investigated to evaluate the CTC/DTC prognostic value in EC. This study included 77 EC patients with complete surgical tumor resection. CTC and DTC were analyzed in blood and bone marrow using nested CK20 reverse transcription-nested polymerase chain reaction (RT-PCR) and findings were correlated with clinical data. Twenty-seven of 76 patients (36.5%) showed CK20 positivity in the blood, 19 of 61 patients (31.1%) in bone marrow, and 40 (51.9%) of 77 patients were positive in either blood or bone marrow or both. In multivariate analyses, only the DTC status emerged as independent predictor of overall and tumor specific survival. Our study revealed that, while the presence of CTC in blood is not associated with a worse prognosis, DTC detection in the bone marrow is a highly specific and independent prognostic marker in EC patients. Larger cohort studies could unravel how this finding can be translated into improved therapy management in EC.

&lt;p&gt;&lt;em&gt;S1PR2&lt;/em&gt; Knockdown Promotes Migration and Invasion in Multiple Myeloma Cells via NF-κB Activation&lt;/p&gt;

BackgroundThe presence of circulating plasma cells (cPCs) was associated with a worse prognosis in multiple myeloma patients. However, the underlying mechanisms involved in the migration and invasion of bone marrow myeloma cells (BMMCs) to cPCs remains unclear. Here, we investigate the possible factors related to hematogenous myeloma cell dissemination and potential regulatory mechanisms.MethodsBMMCs and cPCs of five extramedullary plasmacytoma (EMP) patients were selected for single cell RNA sequencing, We found that the expression level of sphingosine-1-phosphate receptor 2 (S1RP2) was lower in cPCs compared with that in BMMCs. Then, we investigated the effect of S1PR2 in cell migration and invasion through pharmacologic inhibition with a S1PR2-selective antagonist JTE-013 or knockdown of S1PR2 expression in MM cell line U266.ResultsThe results showed that S1PR2 inhibition with JTE-013 or S1PR2-shRNA significantly promoted cell migration and invasion in U266 cells. We measured the expression of invasion-related proteins by Western blot and found that knockdown of S1PR2 could reduce MMP-9 expression in U266 cells. Furthermore, we found NF-κB pathway may mediate the inhibition effects of S1PR2 on cell migration and invasion in MM cells.ConclusionOur findings demonstrated that S1PR2 downregulation may contribute to the initial extramedullary translocation by promoting cell migration and invasion through NF-κB pathway activation.

Observations on Solitary Versus Multiple Isolated Pancreatic Metastases of Renal Cell Carcinoma: Another Indication of a Seed and Soil Mechanism?

Isolated pancreas metastases are a rare type of metastasis of renal cell carcinoma, characterized by the presence of pancreatic metastases, while all other organs remain unaffected. In a previous study, we determined arguments from the literature which (a) indicate a systemic–haematogenic metastasis route (uniform distribution of the metastases across the pancreas and independence of the metastatic localization in the pancreas of the side of the renal carcinoma); and (b) postulate a high impact of a seed and soil mechanism (SSM) on isolated pancreatic metastasis of renal cell carcinoma (isPM) as an explanation for exclusive pancreatic metastases, despite a systemic haematogenous tumor cell embolization. The objective of the study presented was to search for further arguments in favor of an SSM with isPM. For that purpose, the factor’s histology, grading, and singular/multiple pancreas metastases were analyzed on the basis of 814 observations published up to 2018. While histology and grading allowed for no conclusions regarding the importance of an SSM, the comparison of singular/multiple pancreas metastases produced arguments in favor of an SSM: 1. The multiple pancreas metastases observed in 38.1% prove that multiple tumor cell embolisms occur with isPM, the exclusive “maturation” of which in the pancreas requires an SSM; 2. The survival rates (SVR), which are consistent with singular and multiple pancreas metastases (despite the higher total tumor load with the latter), prove that the metastasized tumor cells are not able to survive in all other organs because of an SSM, which results in identical SVR when the pancreatic foci are treated adequately.

Intravascular innate immune cells reprogrammed via intravenous nanoparticles to promote functional recovery after spinal cord injury

Traumatic primary spinal cord injury (SCI) results in paralysis below the level of injury and is associated with infiltration of hematogenous innate immune cells into the injured cord. Methylprednisolone has been applied to reduce inflammation following SCI, yet was discontinued due to an unfavorable risk-benefit ratio associated with off-target effects. In this study, i.v. administered poly(lactide-coglycolide) nanoparticles were internalized by circulating monocytes and neutrophils, reprogramming these cells based on their physicochemical properties and not by an active pharmaceutical ingredient, to exhibit altered biodistribution, gene expression, and function. Approximately 80% of nanoparticle-positive immune cells were observed within the injury, and, additionally, the overall accumulation of innate immune cells at the injury was reduced 4-fold, coinciding with down-regulated expression of proinflammatory factors and increased expression of antiinflammatory and proregenerative genes. Furthermore, nanoparticle administration induced macrophage polarization toward proregenerative phenotypes at the injury and markedly reduced both fibrotic and gliotic scarring 3-fold. Moreover, nanoparticle administration with the implanted multichannel bridge led to increased numbers of regenerating axons, increased myelination with about 40% of axons myelinated, and an enhanced locomotor function (score of 6 versus 3 for control group). These data demonstrate that nanoparticles provide a platform that limits acute inflammation and tissue destruction, at a favorable risk-benefit ratio, leading to a proregenerative microenvironment that supports regeneration and functional recovery. These particles may have applications to trauma and potentially other inflammatory diseases.