Treatment of breast cancer stem cells with oncolytic herpes simplex virus

Cancer stem cells have recently been isolated from several different solid tumors. In breast cancer, the $CD44^{+} CD24^{−/low}$ population is considered to comprise stem-like cells. The identification of cancer stem cells has provided new targets for the development of therapeutics. Oncolytic herpes simplex viruses (oHSVs) are an effective strategy for killing breast cancer cells and treating breast tumors in preclinical models. Here, we examined the efficacy of the oHSV G47Δ in killing breast cancer stem cells. Human breast cancer cell line SK-BR-3 and human primary breast cancer cells were cultured in suspension under conditions conducive to the growth of stem cells. They generated mammospheres, which had cancer stem cell properties. The proportion of $CD44^{+} CD24^{−/low}$ cells in these mammospheres exceeded 95%, as determined by flow cytometry. The mammospheres were found to be highly tumorigenic when implanted subcutaneously in nude BALB/c mice. G47Δ contains the LacZ gene, and X-gal staining of infected cells in vitro and in vivo showed the replication and spread of the virus. G47Δ was found to be highly cytotoxic to the $CD44^{+} CD24^{−/low}$ population in vitro, even when injected at low multiplicities of infection, and G47Δ treatment in vivo significantly inhibited tumor growth compared with mock treatment. This study demonstrates that oHSV is effective against breast cancer stem cells and could be a beneficial strategy for treating breast cancer patients

The transcription factor RUNX2 regulates receptor tyrosine kinase expression in melanoma.

Receptor tyrosine kinases-based autocrine loops largely contribute to activate the MAPK and PI3K/AKT pathways in melanoma. However, the molecular mechanisms involved in generating these autocrine loops are still largely unknown. In the present study, we examine the role of the transcription factor RUNX2 in the regulation of receptor tyrosine kinase (RTK) expression in melanoma. We have demonstrated that RUNX2-deficient melanoma cells display a significant decrease in three receptor tyrosine kinases, EGFR, IGF-1R and PDGFRβ. In addition, we found co-expression of RUNX2 and another RTK, AXL, in both melanoma cells and melanoma patient samples. We observed a decrease in phosphoAKT2 (S474) and phosphoAKT (T308) levels when RUNX2 knock down resulted in significant RTK down regulation. Finally, we showed a dramatic up regulation of RUNX2 expression with concomitant up-regulation of EGFR, IGF-1R and AXL in melanoma cells resistant to the BRAF V600E inhibitor PLX4720. Taken together, our results strongly suggest that RUNX2 might be a key player in RTK-based autocrine loops and a mediator of resistance to BRAF V600E inhibitors involving RTK up regulation in melanoma

Cancer Stem Cell-Like Cells Derived from Malignant Peripheral Nerve Sheath Tumors

This study aims to examine whether or not cancer stem cells exist in malignant peripheral nerve sheath tumors (MPNST). Cells of established lines, primary cultures and freshly dissected tumors were cultured in serum free conditions supplemented with epidermal and fibroblast growth factors. From one established human MPNST cell line, S462, cells meeting the criteria for cancer stem cells were isolated. Clonal spheres were obtained, which could be passaged multiple times. Enrichment of stem cell-like cells in these spheres was also supported by increased expression of stem cell markers such as CD133, Oct4, Nestin and NGFR, and decreased expression of mature cell markers such as CD90 and NCAM. Furthermore, cells of these clonal S462 spheres differentiated into Schwann cells, smooth muscle/fibroblast and neurons-like cells under specific differentiation-inducing cultural conditions. Finally, subcutaneous injection of the spheres into immunodeficient nude mice led to tumor formation at a higher rate compared to the parental adherent cells (66% versus 10% at 2.5×105). These results provide evidence for the existence of cancer stem cell-like cells in malignant peripheral nerve sheath tumors

Reconstructing and Reprogramming the Tumor-Propagating Potential of Glioblastoma Stem-like Cells

Developmental fate decisions are dictated by master transcription factors (TFs) that interact with cis-regulatory elements to direct transcriptional programs. Certain malignant tumors may also depend on cellular hierarchies reminiscent of normal development but superimposed on underlying genetic aberrations. In glioblastoma (GBM), a subset of stem-like tumor-propagating cells (TPCs) appears to drive tumor progression and underlie therapeutic resistance, yet remain poorly understood. Here, we identify a core set of neurodevelopmental TFs (POU3F2, SOX2, SALL2, OLIG2) essential for GBM propagation. These TFs coordinately bind and activate TPC-specific regulatory elements, and are sufficient to fully reprogram differentiated GBM cells to ‘induced’ TPCs, recapitulating the epigenetic landscape and phenotype of native TPCs. We reconstruct a network model that highlights critical interactions and identifies novel therapeutic targets for eliminating TPCs. Our study establishes the epigenetic basis of a developmental hierarchy in GBM, provides detailed insight into underlying gene regulatory programs, and suggests attendant therapeutic strategies

Integrated genomic characterization of oesophageal carcinoma

Oesophageal cancers are prominent worldwide; however, there are few targeted therapies and survival rates for these cancers remain dismal. Here we performed a comprehensive molecular analysis of 164 carcinomas of the oesophagus derived from Western and Eastern populations. Beyond known histopathological and epidemiologic distinctions, molecular features differentiated oesophageal squamous cell carcinomas from oesophageal adenocarcinomas. Oesophageal squamous cell carcinomas resembled squamous carcinomas of other organs more than they did oesophageal adenocarcinomas. Our analyses identified three molecular subclasses of oesophageal squamous cell carcinomas, but none showed evidence for an aetiological role of human papillomavirus. Squamous cell carcinomas showed frequent genomic amplifications of CCND1 and SOX2 and/or TP63, whereas ERBB2, VEGFA and GATA4 and GATA6 were more commonly amplified in adenocarcinomas. Oesophageal adenocarcinomas strongly resembled the chromosomally unstable variant of gastric adenocarcinoma, suggesting that these cancers could be considered a single disease entity. However, some molecular features, including DNA hypermethylation, occurred disproportionally in oesophageal adenocarcinomas. These data provide a framework to facilitate more rational categorization of these tumours and a foundation for new therapies

Center-Level Variation in Transplant Rates Following the Heart Allocation Policy Change.

Importance: Wide state-level variability in waiting list outcomes have been noted for patients listed for heart transplant in the US, but little is known regarding center-level transplant rates since the heart allocation policy change. Objective: To evaluate center-level transplant rates following the recent allocation policy change for heart transplant. Design, Setting, and Participants: This cohort study used data from the United Network for Organ Sharing database from October 18, 2015, to March 1, 2020, for a nationwide analysis of transplant centers in the US. Transplant candidates were stratified into 2 time cohorts, with era 1 denoting the 3-year period before the policy change (October 18, 2018), and era 2 representing the 500-day period after the policy change but before the beginning of the COVID-19 pandemic. Data were analyzed from May to June 2021. Exposure: The heart allocation policy change enacted on October 18, 2018. Main Outcomes and Measures: Competing risk regression for waiting list outcomes was performed to calculate adjusted era 1 and era 2 center-level transplant rates. Rates were compared across regions and states, as well as within organ procurement organizations. Pearson correlation coefficient was used to assess center-level factors associated with era 2 transplant rates. Results: Of 15 940 transplant candidates included for analysis, 5063 (median [IQR] age, 56 [45-63] years; 1385 women [27.4%]) comprised the era 2 cohort. The proportion of patients with temporary mechanical circulatory support increased between era 1 and era 2 (extracorporeal membrane oxygenation, 2.00% vs 3.42%; percutaneous ventricular assist device, 0.66% vs 1.86%; intra-aortic balloon pump, 5.21% vs 13.10%). The adjusted mean center-level likelihood of transplant increased after the rule change (from 48.1% in era 1 to 78.0% in era 2). Significant variation in transplant rates was observed across regions and states even among centers with shared organ procurement organizations. The largest absolute difference in transplant rates was 27.1% for 2 centers belonging to the same organ procurement organization. Centers with higher transplant volumes in era 2 and with a greater proportion of candidates with intra-aortic balloon pump were observed to have higher transplant rates. Conclusions and Relevance: Despite sharing organ supply and having a small geographical distance, these findings suggest that intercenter disparities in the likelihood of transplant have persisted following the heart allocation policy change. Further work is necessary to ensure equitable allocation of organs in heart transplant

Outcomes of coronary artery bypass surgery using modified del Nido cardioplegia in patients with poor ventricular function

Abstract Background del Nido cardioplegia (DN) has been shown to be safe in adult patients undergoing isolated coronary artery bypass grafting with normal left ventricular ejection fraction. We sought to determine whether it was also safe in adult patients with diminished left ventricular function. Methods All patients with preoperative left ventricular ejection fraction ≤ 40% undergoing isolated coronary artery bypass grafting between 1/1/2019 and 7/10/2022 were retrospectively analyzed. Off-pump and beating heart cases were excluded. Patients were divided by surgeon preference between conventional cardioplegia (CCP) and DN. Baseline and intraoperative characteristics and short-term postoperative outcomes were compared. Results Six surgeons performed 829 isolated coronary artery bypass operations during the study. Two-hundred seventy-two met study criteria. Three surgeons used exclusively CCP for the duration of the study, two used exclusively DN and one switched from CCP to DN mid-way through. Group totals were: CCP n = 181 and DN n = 91. There were no significant differences in baseline characteristics including mean left ventricular ejection fraction (CCP 32.5 ± 7.4% vs. DN 33.4 ± 7.29%, p = 0.939). Other than a significant decrease in bypass time for DN (113.20 ± 37.2 vs. 122.43 ± 34.3 min, p = 0.043) there were no intergroup differences in urgency, number of grafts, ischemic time or incidence of blood transfusion. Postoperative outcomes between CCP and DN were similar including incidence of atrial fibrillation (12.2% vs. 8.8%, p = 0.403), intensive care length of stay (3.7 ± 2.3 vs. 4.3 ± 3.7 days, p = 0.886), total length of stay (5.7 ± 3.7 vs. 6.3 ± 4.4 days, p = 0.922) and 30-day mortality (3.85% vs. 1.10%, p = 0.205). Conclusion Compared to conventional cardioplegia, del Nido cardioplegia provides equivalent short-term outcomes in patients with low left ventricular ejection fraction undergoing isolated coronary artery bypass grafting

Multi-lineage differentiation of cells of clonal S462 spheres.

<p>Adherent cells (left column) and dissociated sphere cells (right columns) were cultured with growth factors inducing differentiation into cells resembling (A) Schwann cells, (B) SM/Fb and (C) neurons, which are positively stained for S100/NGFR/neurofilament (Schwann cells), SMA (SM/Fb), and MAP-2 (neurons), respectively. Insert in A illustrates S100+ Schwann cells derived from a plexiform neurofibroma culture. (D) phase contrast micrographs from differentiated cells under 3 culture conditions to generate Schwann cell, SM/Fb and neuron-like cells. Differentiated cells are indicated by arrows and non-differentiated cells by arrowheads. Bars = 20 µm. NGFR, nerve growth factor; SMA, smooth muscle actin; MAP-2, microtubule associated protein-2; PNF, plexiform neurofibroma; LC, like-cells.</p

Co-expression of NGFR and CD133 in adherent and S462 spheres.

<p>Side and forward scatter analysis of adherent cells (left column) and spheres (at passage 8, right column) showed different types of cells. Live cells were gated in R1 (upper panels), and gatings for NGFR (R2) positive cells (middle panels) are shown in orange. NGFR-positive cells (represented in orange) within the CD133-positive cells show co-expression of CD133/NGFR (lower panels). NGFR cells co-expressing CD133 are infrequent in the adherent cell population and increased in spheres. Isotype controls for all the antigens were used to set up the quadrants for negative populations. NGFR, nerve growth factor receptor.</p

Proliferation and self-renewal of MPNST spheres.

<p>Bromodeoxyuridine incorporation assay revealed proliferation of S462 cells in the clonal spheres (secondary spheres). The increase in cell absorbance was significant for 8, 10, and 15 days in SCM versus 3 days (p<0.01 **). Changes in the frequency of sphere formation in wells containing single cells originally from adherent S462, then from dissociated spheres plated as single cells and passaged consecutively. The increase in frequency of sphere formation with increasing passage was significant (passage 1 (primary spheres) versus passages 10 (10^th spheres) and 12 (12^th spheres), P<0.01 **, passage 2 (secondary spheres) versus passages 10 (10^th spheres) and 12^th spheres), P<0.05*).</p