Neuroendocrine tumors (NETs) occur primarily in the small intestine, lung, and pancreas. Due to their rarity compared to other malignancies in these organs, their complex biology remains poorly understood, including their oncogenesis, tumor composition, and the intriguing phenomena of mixed neuroendocrine non-neuroendocrine neoplasms (MiNEN). Here, we profiled ten low-grade small intestine NET (SiNET) samples as well as one mixed lung tumor by single-cell or single-nuclei RNA-seq. We find that SiNETs are largely separated into two distinct subtypes, in which the neuroendocrine cells upregulate epithelial or neuronal markers, respectively. Surprisingly, in both subtypes, the neuroendocrine cells are largely non-proliferative while higher proliferation is observed in multiple non-malignant cell types. Specifically, B and plasma cells are highly proliferative in the epithelial-like SiNET subtype, potentially reflecting the outcome of high Migration Inhibitory Factor (MIF) expression in those tumors, which may constitute a relevant target. Finally, our analysis of a mixed lung neuroendocrine tumor identifies a population of putative progenitor cells that may give rise to both neuroendocrine and non-neuroendocrine (squamous) cells, potentially explaining the origin of the mixed histology. Taken together, our results provide important insights and hypotheses regarding the biology of neuroendocrine neoplasms.

Each tumour contains diverse cellular states that underlie intratumour heterogeneity (ITH), a central challenge of cancer therapeutics(1). Dozens of recent studies have begun to describe ITH by single-cell RNA sequencing, but each study typically profiled only a small number of tumours and provided a narrow view of transcriptional ITH2. Here we curate, annotate and integrate the data from 77 different studies to reveal the patterns of transcriptional ITH across 1,163 tumour samples covering 24 tumour types. Among the malignant cells, we identify 41 consensus meta-programs, each consisting of dozens of genes that are coordinately upregulated in subpopulations of cells within many tumours. The meta-programs cover diverse cellular processes including both generic (for example, cell cycle and stress) and lineage-specific patterns that we map into 11 hallmarks of transcriptional ITH. Most meta-programs of carcinoma cells are similar to those identified in non-malignant epithelial cells, suggesting that a large fraction of malignant ITH programs are variable even before oncogenesis, reflecting the biology of their cell of origin. We further extended the meta-program analysis to six common non-malignant cell types and utilize these to map cell-cell interactions within the tumour microenvironment. In summary, we have assembled a comprehensive pan-cancer single-cell RNA-sequencing dataset, which is available through the Curated Cancer Cell Atlas website, and leveraged this dataset to carry out a systematic characterization of transcriptional ITH.

Cell migration is a key step of cancer metastasis, immune-cell navigation, homing of stem cells and development. What adds complexity to it is the heterogeneity of the tissue environment that gives rise to a vast diversity of migratory mechanisms utilized by cells. A majority of cell motility mechanisms reported elsewhere largely converge in depicting the importance of the activity and complexity of actomyosin networks in the cell. In this review, we highlight the less discussed functional diversity of these actomyosin complexes and describe in detail how the major cellular actin-binding molecular motor proteins, nonmuscle myosin IIs are regulated and how they participate and mechanically reciprocate to changes in the microenvironment during cancer cell migration and tumor progression. Understanding the role of nonmuscle myosin IIs in the cancer cell is important for designing efficient therapeutic strategies to prevent cancer metastasis.

Each tumor contains malignant cells that differ in genotype, phenotype, and in their interactions with the tumor micro-environment (TME). This results in distinct integrated cellular states that govern intra-tumor heterogeneity (ITH), a central challenge of cancer therapeutics. Dozens of recent studies have begun to describe ITH by single cell RNA-seq, but each study typically profiledonly a small number of tumors and provided a narrow view of transcriptional ITH. Here, we curate, annotate and integrate the data from 77 different studies to reveal the patterns of ITH across 1,163 tumor samples covering 24 tumor types. Focusing on the malignant cells, we find thousands of transcriptional ITH programs that can be described by 41 consensus meta-programs (MPs), each consisting of dozens of genes that are coordinately upregulated in subpopulations of cells within many different tumors. The MPs cover diverse cellular processes and differ in their cancer-type distribution. General MPs associated with processes such as cell cycle and stress vary within most tumors, while context-specific MPs reflect the unique biology of particular cancer types, often resembling developmental cell types and suggesting the co-existence of variable differentiation states within tumors. Some of the MPs are further associated with overall tumor proliferation or immune state, highlighting their potential clinical significance. Based on functional similarities among MPs, we propose a set of 11 hallmarks that together account for the majority of observed ITH programs. Given the breadth and scope of the investigated cohort, the MPs and hallmarks described here reflect the first comprehensive pan-cancer description of transcriptional ITH.

Though many cancers are known to show up-regulation of nonmuscle myosin (NM) IIA and IIB, the mechanism by which NMIIs aid in cancer development remains unexplored. Here we demonstrate that tumor-generating, fibroblast-like cells isolated from 3-methylcholanthrene (3MC)-induced murine tumor exhibit distinct phospho-dependent localization of NMIIA and NMIIB at the perinuclear area and tip of the filopodia and affect cell migration differentially. While NMIIA-KD affects protrusion dynamics and increases cell directionality, NMIIB-KD lowers migration speed and increases filopodial branching. Strategically located NMIIs at the perinuclear area colocalize with the linker of nucleoskeleton and cytoskeleton (LINC) protein Nesprin2 and maintain the integrity of the nuclear-actin cap. Interestingly, knockdown of NMIIs results in altered expression of genes involved in epithelial-to-mesenchymal transition, angiogenesis, and cellular senescence. NMIIB-KD cells display down-regulation of Gsc and Serpinb2, which is strikingly similar to Nesprin2-KD cells as assessed by quantitative PCR analysis. Further gene network analysis predicts that NMIIA and NMIIB may act on similar pathways but through different regulators. Concomitantly, knockdown of NMIIA or NMIIB lowers the growth rate and tumor volume of 3MC-induced tumor in vivo. Altogether, these results open a new window to further investigate the effect of LINC-associated perinuclear actomyosin complex on mechanoresponsive gene expression in the growing tumor.

Size and structural oscillation of the non-muscle myosin (NM) II-C2 protein are investigated using time resolved confocal microscopy. It is observed that the high enzymatic activity of GFP tagged NM II-C2 is invariably accompanied by frequent fluctuations in the fluorescence intensity (fluorescence oscillation) of GFP in Neuro-2a cells. We demonstrate that the deletion of the polar rich N-terminal 12 amino acid (aa) of the C2 insert diminishes the fluorescence oscillation of NM II-C2. On deletion of either the middle 21 aa or the C-terminal 8 aa, frequency of observing periodic fluctuation in fluorescence intensity decreases. Fluorescence correlation spectroscopy (FCS) was used to monitor the size of the GFP tagged NM II-C2. NM II-C2-GFP molecule remains in the rod-like extended conformation (6S) with a length of 120 +/- 5 nm while deletion of any region of the C2 insert causes the molecule to adopt the 10S conformation with a length of 50-65 nm. These findings suggest that enzymatic activity and assembly property of NM II-C2 are controlled by the polar amino acids present in the C2 insert.

In this study, we investigated the regions in the alternatively spliced C2 insert of nonmuscle myosin (NM) II‐C conferring unique functional properties to the protein. We used constructs carrying deletions within different regions of C2 in neuronal cells; namely, the polar N terminus, the proline/serine‐rich middle, and the nonpolar C terminus. We compared the wild‐type NM II‐C2 and deletion mutants with respect to ATPase activity, coassembly with NM II‐B, regulation by myosin light‐chain kinase (MLCK), and solubility, to determine the C2 region(s) involved in these processes. In addition, we examined the ability of the mutants to rescue the neurite‐shortening phenotype upon NM II‐C2 knockdown in Neuro‐2a cells. Our data highlight the importance of the polar N terminus in NM II‐C2 function.

Front Cover: The cover shows the mechanism of a SRN1-polymerization for formation of industrially relevant poly(paraphenylenesulfide) (PPS). On the left side, the lamp represents the irradiation source of this light-initiated polymerization process in front of a reaction flask. Further details can be found in the article by N. B. Heine, and A. Studer* on page 1494.

The synthesis, micellar aggregation, and pH-triggered intracellular drug delivery ability of an amphiphilic statistical copolymer (P2) are studied. Two methacrylate derivatives, one containing a hydrophilic pendant and the other containing a hydrophobic pendant chain, are copolymerized to produce P2. The hydrophobic pendant chain is linked to the polymer backbone by a β-thiopropionate linkage, known to undergo slow hydrolysis at mild acidic pH. P2 forms a multimicellar cluster in water with a critical aggregation concentration of 0.02 mg mL(-1) and encapsulates a hydrophobic guest such as pyrene, Nile red, or the anti-cancer drug doxorubicin (Dox). Sustained release of the entrapped Dox (80% after 100 h) is noticed at pH 5.2, while release is significantly slower (35% after 100 h) at pH 7.4. Acidic hydrolysis of the β-thiopropionate linkage leading to the reduction of the hydrophobicity is established as the cause for micellar disassembly and triggered drug release. Cell-culture studies with the human breast cancer cell line, MCF-7, reveal biocompatibility of P2 (below 150 μg mL(-1) ). It is further tested for intracellular delivery of Dox. MCF-7 cells remain healthy at pH 7.4 but become unhealthy at pH 5.2 when treated with a Dox-loaded P2 micelles.

Previous studies reported that inclusion of 41 aa (33 aa in human) insert in loop 2 of mouse nonmuscle myosin (NM) II‐C heavy chain makes the molecule to become independent of its regulatory light chain (RLC) phosphorylation in MgATPase activity and in vitro motility, and to localize in the neurites as puncta form. However, the molecular mechanism behind these unique properties among myosin II family is not known. Here, we show that deletion of the N‐terminal 12 aa, but not the C‐terminal 8 aa or the middle 21 aa of C2 insert, diminishes the fluorescence oscillation property of GFP tagged‐NM II‐C2 molecule in the neurites of neuro‐2a cells as determined by fluorescence lifetime imaging microscopy analysis. Also, we demonstrate that inclusion of 41 aa insert increases the population of extended monomers whereas deletion of N‐terminal 12 aa, middle 21 aa or C‐terminal 8 aa of 41 aa insert increases the folded autoinhibited NM II‐C2 monomers as revealed by fluorescence correlation spectroscopy (FCS) analysis in neuronal cells. Furthermore, FCS analysis reveals that NM II‐Δ12C2‐GFP significantly diffuses very slowly compared with NM II‐Δ8C2‐ or NM II‐Δ21C2‐GFP in the neurites, suggesting that radius (rH) of NM II‐Δ12 C2‐GFP is higher than that of wildtype or mutants Δ21 and Δ8C2‐GFP. These studies suggest that N‐terminal 12 aa (contributor of net charge of C2) plays a significant role in determining the oscillation and aggregation properties of the molecule.Research Support: Department of Science and Technology, India