Lung Cancer Induces NK Cell Contractility and Cytotoxicity Through Transcription Factor Nuclear Localization.

Actomyosin-mediated cellular contractility is highly conserved for mechanotransduction and signalling. While this phenomenon has been observed in adherent cell models, whether/how contractile forces regulate the function of suspension cells like natural killer (NK) cells during cancer surveillance, is unknown. Here, we demonstrated in coculture settings that the evolutionarily conserved NK cell transcription factor, Eomes, undergoes nuclear shuttling during lung cancer cell surveillance. Biophysical and biochemical analyses revealed mechanistic enhancement of NK cell actomyosin-mediated contractility, which is associated with nuclear flattening, thus enabling nuclear entry of Eomes associated with enhanced NK cytotoxicity. We found that NK cells responded to the presumed immunosuppressive TGFß in the NK-lung cancer coculture medium to sustain its intracellular contractility through myosin light chain phosphorylation, thereby promoting Eomes nuclear localization. Therefore, our results demonstrate that lung cancer cells provoke NK cell contractility as an early phase activation mechanism and that Eomes is a plausible mechano-responsive protein for increased NK cytotoxicity. There is scope for strategic application of actomyosin-mediated contractility modulating drugs ex vivo, to reinvigorate NK cells prior to adoptive cancer immunotherapy in vivo (177 words).

pHLuc, a Ratiometric Luminescent Reporter for in vivo Monitoring of Tumor Acidosis.

Even under normoxia, cancer cells exhibit increased glucose uptake and glycolysis, an occurrence known as the Warburg effect. This altered metabolism results in increased lactic acid production, leading to extracellular acidosis and contributing to metastasis and chemoresistance. Current pH imaging methods are invasive, costly, or require long acquisition times, and may not be suitable for high-throughput pre-clinical small animal studies. Here, we present a ratiometric pH-sensitive bioluminescence reporter called pHLuc for in vivo monitoring of tumor acidosis. pHLuc consists of a pH-sensitive GFP (superecliptic pHluorin or SEP), a pH-stable OFP (Antares), and Nanoluc luciferase. The resulting reporter produces a pH-responsive green 510nm emission (from SEP) and a pH-insensitive red-orange 580nm emission (from Antares). The ratiometric readout (R580 / 510) is indicative of changes in extracellular pH (pHe). In vivo proof-of-concept experiments with NSG mice model bearing human synovial sarcoma SW982 xenografts that stably express the pHLuc reporter suggest that the level of acidosis varies across the tumor. Altogether, we demonstrate the diagnostic value of pHLuc as a bioluminescent reporter for pH variations across the tumor microenvironment. The pHLuc reporter plasmids constructed in this work are available from Addgene.

Macrophages protect mycoplasma-infected chronic myeloid leukemia cells from natural killer cell killing.

Macrophages (Mϕ) have been reported to downmodulate the cytotoxicity of natural killer (NK) cell against solid tumor cells. However, the collaborative role between NK cells and Mϕ remains underappreciated, especially in hematological cancers, such as chronic myeloid leukemia (CML). We observed a higher ratio of innate immune cells (Mϕ and NK) to adaptive immune cells (T and B cells) in CML bone marrow aspirates, prompting us to investigate the roles of NK and Mϕ in CML. Using coculture models simulating the tumor inflammatory environment, we observed that Mϕ protects CML from NK attack only when CML was itself mycoplasma-infected and under chronic infection-inflammation condition. We found that the Mϕ-protective effect on CML was associated with the maintenance of CD16 level on the NK cell membrane. Although the NK membrane CD16 (mCD16) was actively shed in Mϕ + NK + CML trioculture, the NK mCD16 level was maintained, and this was independent of the modulation of sheddase by tissue inhibitor of metalloproteinase 1 or inhibitory cytokine transforming growth factor beta. Instead, we found that this process of NK mCD16 maintenance was conferred by Mϕ in a contact-dependent manner. We propose a new perspective on anti-CML strategy through abrogating Mϕ-mediated retention of NK surface CD16.

Novel AU-rich proximal UTR sequences (APS) enhance CXCL8 synthesis upon the induction of rpS6 phosphorylation.

The role of ribosomal protein S6 (rpS6) phosphorylation in mRNA translation remains poorly understood. Here, we reveal a potential role in modulating the translation rate of chemokine (C-X-C motif) ligand 8 (CXCL8 or Interleukin 8, IL8). We observed that more CXCL8 protein was being secreted from less CXCL8 mRNA in primary macrophages and macrophage-like HL-60 cells relative to other cell types. This correlated with an increase in CXCL8 polyribosome association, suggesting an increase in the rate of CXCL8 translation in macrophages. The cell type-specific expression levels were replicated by a CXCL8- UTR-reporter (Nanoluc reporter flanked by the 5' and 3' UTR of CXCL8). Mutations of the CXCL8-UTR-reporter revealed that cell type-specific expression required: 1) a 3' UTR of at least three hundred bases; and 2) an AU base content that exceeds fifty percent in the first hundred bases of the 3' UTR immediately after the stop codon, which we dub AU-rich proximal UTR sequences (APS). The 5' UTR of CXCL8 enhanced expression at the protein level and conferred cell type-specific expression when paired with a 3' UTR. A search for other APS-positive mRNAs uncovered TNF alpha induced protein 6 (TNFAIP6), another mRNA that was translationally upregulated in macrophages. The elevated translation of APS-positive mRNAs in macrophages coincided with elevated rpS6 S235/236 phosphorylation. Both were attenuated by the ERK1/2 signaling inhibitors, U0126 and AZD6244. In A549 cells, rpS6 S235/236 phosphorylation was induced by TAK1, Akt or PKA signaling. This enhanced the translation of the CXCL8-UTR-reporters. Thus, we propose that the induction of rpS6 S235/236 phosphorylation enhances the translation of mRNAs that contain APS motifs, such as CXCL8 and TNFAIP6. This may contribute to the role of macrophages as the primary producer of CXCL8, a cytokine that is essential for immune cell recruitment and activation.