IL1R1+ cancer-associated fibroblasts drive tumor development and immunosuppression in colorectal cancer.

Fibroblasts have a considerable functional and molecular heterogeneity and can play various roles in the tumor microenvironment. Here we identify a pro-tumorigenic IL1R1+, IL-1-high-signaling subtype of fibroblasts, using multiple colorectal cancer (CRC) patient single cell sequencing datasets. This subtype of fibroblasts is linked to T cell and macrophage suppression and leads to increased cancer cell growth in 3D co-culture assays. Furthermore, both a fibroblast-specific IL1R1 knockout and IL-1 receptor antagonist Anakinra administration reduce tumor growth in vivo. This is accompanied by reduced intratumoral Th17 cell infiltration. Accordingly, CRC patients who present with IL1R1-expressing cancer-associated-fibroblasts (CAFs), also display elevated levels of immune exhaustion markers, as well as an increased Th17 score and an overall worse survival. Altogether, this study underlines the therapeutic value of targeting IL1R1-expressing CAFs in the context of CRC.

Cell type-dependent ROS and mitophagy response leads to apoptosis or necroptosis in neuroblastoma.

A limiting factor in the therapeutic outcome of children with high-risk neuroblastoma is the intrinsic and acquired resistance to common chemotherapeutic treatments. Here we investigated the molecular mechanisms by which the hemisynthetic cardiac glycoside UNBS1450 overcomes this limitation and induces differential cell death modalities in both neuroblastic and stromal neuroblastoma through stimulation of a cell-type-specific autophagic response eventually leading to apoptosis or necroptosis. In neuroblastic SH-SY5Y cells, we observed a time-dependent production of reactive oxygen species that affects lysosomal integrity inducing lysosome-associated membrane protein 2 degradation and cathepsin B and L activation. Subsequent mitochondrial membrane depolarization and accumulation of mitochondria in phagophores occurred after 8h of UNBS1450 treatment. Results were confirmed by mitochondrial mass analysis, electron microscopy and co-localization of mitochondria with GFP-LC3, suggesting the impaired clearance of damaged mitochondria. Thus, a stress-induced defective autophagic flux and the subsequent lack of clearance of damaged mitochondria sensitized SH-SY5Y cells to UNBS1450-induced apoptosis. Inhibition of autophagy with small inhibitory RNAs against ATG5, ATG7 and Beclin-1 protected SH-SY5Y cells against the cytotoxic effect of UNBS1450 by inhibiting apoptosis. In contrast, autophagy progression towards the catabolic state was observed in stromal SK-N-AS cells: here reactive oxygen species (ROS) generation remained undetectable preserving intact lysosomes and engulfing damaged mitochondria after UNBS1450 treatment. Moreover, autophagy inhibition determined sensitization of SK-N-AS to apoptosis. We identified efficient mitophagy as the key mechanism leading to failure of activation of the apoptotic pathway that increased resistance of SK-N-AS to UNBS1450, triggering rather necroptosis at higher doses. Altogether we characterize here the differential modulation of ROS and mitophagy as a main determinant of neuroblastoma resistance with potential relevance for personalized anticancer therapeutic approaches.

Early downregulation of Mcl-1 regulates apoptosis triggered by cardiac glycoside UNBS1450.

Cardiac glycosides (CGs), prescribed to treat cardiovascular alterations, display potent anti-cancer activities. Despite their well-established target, the sodium/potassium (Na(+)/K(+))-ATPase, downstream mechanisms remain poorly elucidated. UNBS1450 is a hemi-synthetic cardenolide derived from 2″-oxovorusharin extracted from the plant Calotropis procera, which is effective against various cancer cell types with an excellent differential toxicity. By comparing adherent and non-adherent cancer cell types, we validated Mcl-1 as a general and early target of UNBS1450. A panel of CGs including cardenolides ouabain, digitoxin and digoxin as well as bufadienolides cinobufagin and proscillaridin A allowed us to generalize our findings. Our results show that Mcl-1, but not Bcl-xL nor Bcl-2, is rapidly downregulated prior to induction of apoptosis. From a mechanistic point of view, we exclude an effect on transcription and demonstrate involvement of a pathway affecting protein stability and requiring the proteasome in the early CG-induced Mcl-1 downregulation, without the involvement of caspases or the BH3-only protein NOXA. Strategies aiming at preventing UNBS1450-induced Mcl-1 downregulation by overexpression of a mutated, non-ubiquitinable form of the protein or the use of the proteasome inhibitor MG132 inhibited the compound's ability to induce apoptosis. Altogether our results point at Mcl-1 as a ubiquitous factor, downregulated by CGs, whose modulation is essential to achieve cell death.

A new dimension for cell identification by FTIR spectroscopy: depth profiling in attenuated total reflection.

The multiresistant phenotype is an important problem in cancer chemotherapy. It is characterized by cell resistance to multiple and structurally unrelated drugs. We have shown previously that K562 multiresistant leukemia cells could be differentiated from their sensitive counterparts (wild-type K562 cells) on the basis of their infrared spectrum. In ATR FTIR mode, the penetration depth is controlled by both the wavelength and the incident angle, allowing depth profiling of samples. In this paper we took advantage of the ATR capability to modulate the penetration depth of the infrared wave into the cell, by modulating the incident angle, to investigate the differences between K562 multiresistant cells and their sensitive counterparts (wild-type K562 cells) as a function of this penetration depth. It is shown that focusing the IR beam on the most discriminant depth allows improvement of the discrimination between multiresistant and sensitive K562 cells. It is suggested that the depth profile of the difference spectra could allow a more precise localization of the biochemical modifications arising within the multidrug resistance phenotype.

Cell discrimination by attenuated total reflection-Fourier transform infrared spectroscopy: the impact of preprocessing of spectra.

Fourier transform infrared (FT-IR) spectroscopy has become a powerful tool for biodiagnostics and cell line classification. Typical experimental perturbations included in spectra are baseline shift and scale variation between spectra. They have to be removed by data preprocessings to allow further data analysis and classification. In this work, we addressed baseline shift corrections and normalizations in attenuated total reflection (ATR) FT-IR spectra. We compared the efficiency of several preprocessing methods with series of spectra containing typical perturbations (baseline shift, scaling factor, and noise) and a priori known definite spectral difference. Several baseline-correction and normalization possibilities were evaluated. Our results were generally sensitive, selective, and robust with respect to baseline and scaling. Full-range scaling generated more false-positive results. Use of first- and second-derivative spectra was tested. Results obtained on model spectra were confirmed with series of spectra from sensitive and multidrug-resistant leukemia K562 cells. We showed that the use of derived spectra did not provide more efficiency and required additional preprocessing such as smoothing to obtain results similar to those obtained from non-derived ones. On the other hand, results obtained with derivatives were less sensitive to scaling, a useful feature when scaling is problematic.

Effect of the antibiotic azithromycin on thermotropic behavior of DOPC or DPPC bilayers.

Azithromycin is a macrolide antibiotic known to bind to lipids and to affect endocytosis probably by interacting with lipid membranes [Tyteca, D., Schanck, A., Dufrene, Y.F., Deleu, M., Courtoy, P.J., Tulkens, P.M., Mingeot-Leclercq, M.P., 2003. The macrolide antibiotic azithromycin interacts with lipids and affects membrane organization and fluidity: studies on Langmuir-Blodgett monolayers, liposomes and J774 macrophages. J. Membr. Biol. 192, 203-215]. In this work, we investigate the effect of azithromycin on lipid model membranes made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Thermal transitions of both lipids in contact with azithromycin are studied by (31)P NMR and DSC on multilamellar vesicles. Concerning the DPPC, azithromycin induces a suppression of the pretransition whereas a phase separation between the DOPC and the antibiotic is observed. For both lipids, the enthalpy associated with the phase transition is strongly decreased with azithromycin. Such effects may be due to an increase of the available space between hydrophobic chains after insertion of azithromycin in lipids. The findings provide a molecular insight of the phase merging of DPPC gel in DOPC fluid matrix induced by azithromycin [Berquand, A., Mingeot-Leclercq, M.P., Dufrene, Y.F., 2004. Real-time imaging of drug-membrane interactions by atomic force microscopy. Biochim. Biophys. Acta 1664, 198-205] and could help to a better understanding of azithromycin-cell interaction.

The infrared spectrum of human glioma cells is related to their in vitro and in vivo behavior.

The present research investigates whether infrared spectra can be related to the biological characteristics of glioma cell lines. We used nine human glioma cell lines for which a series of in vitro and in vivo biological features had already been established [Glia 36 (2001) 375] and were able to show that their characteristic infrared spectra reflect their in vitro migration (i.e., motility and invasiveness) properties and their in vivo aggressiveness. More particularly, the infrared data evidenced correlations at the level of the lipid/protein ratio. These relationships were found to be tissue-dependent when controlled on seven pancreatic carcinoma cell lines. We also showed that oligodendroglial and astrocytic tumor cells, whose identification remains difficult, can easily be identified by their infrared spectra in the lipid acyl chain region as well as in the nucleic acid region. We concluded that infrared spectroscopy could usefully complement information provided by more conventional diagnostic and prognostic (e.g., morphological and molecular) approaches.