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Abstract The interferon signalling system elicits a robust cytokine response against a wide range of environmental pathogenic and internal pathological signals, leading to induction of a subset of interferon-induced proteins. We applied DSS (disuccinimidyl suberate) mediated cross-linking mass spectrometry (CLMS) to capture novel protein–protein interactions within the realm of interferon induced proteins. In addition to the expected interferon-induced proteins, we identified novel inter- and intra-molecular cross-linked adducts for the canonical interferon induced proteins, such as MX1, USP18, OAS3, and STAT1. We focused on orthogonal validation of a cohort of novel interferon-induced protein networks formed by the HLA-A protein (H2BFS-HLA-A-HMGA1) using co-immunoprecipitation assay, and further investigated them by molecular dynamics simulation. Conformational dynamics of the simulated protein complexes revealed several interaction sites that mirrored the interactions identified in the CLMS findings. Together, we showcase a proof-of-principle CLMS study to identify novel interferon-induced signaling complexes and anticipate broader use of CLMS to identify novel protein interaction dynamics within the tumour microenvironment.

The characteristic mechanical forces at play within tumors include the abnormal solid and fluid stresses. These, together with the increased extracellular matrix (ECM) stiffness, are the major transport barriers affecting the nanomedicine delivery to solid tumors. Due to the elevated pressure within the tumor microenvironment, the transport of nanomedicines through the interstitial space is limited to diffusion. While this particular scenario is central for nanomedicine delivery to solid tumors, it has not been modeled in vitro before. To this end, herein, a tumor‐on‐a‐chip microfluidic device is developed that is capable of recapitulating the solid stress scenario in tumors. This is achieved by integrating a pneumatic actuation to apply compression to the enclosed hydrogel ECM filling medium. Transport studies of model nanoparticles (NPs) across this medium are performed to determine their diffusion. For these NPs, it is demonstrated that their transport is drastically reduced by 65% due to the compression of the ECM gel matrix, reducing its pore size, with only an applied pressure of ≈4 Pa. The results obtained show that the actuated tumor‐on‐a‐chip device can be used to evaluate the diffusive penetration capability of nanomedicines within a mechanical‐constrained microenvironment such that of tumors.

Nrf2 (nuclear factor erythroid 2 (NF-E2)-related factor 2) transcription factor is recognized for its pro-survival and cell protective role upon exposure to oxidative, chemical, or metabolic stresses. Nrf2 controls a number of cellular processes such as proliferation, differentiation, apoptosis, autophagy, lipid synthesis, and metabolism and glucose metabolism and is a target of activation in chronic diseases like diabetes, neurodegenerative, and inflammatory diseases. The dark side of Nrf2 is revealed when its regulation is imbalanced (e.g., via oncogene activation or mutations) and under such conditions constitutively active Nrf2 promotes cancerogenesis, metastasis, and radio- and chemoresistance. When there is no stress, Nrf2 is instantly degraded via Keap1-Cullin 3 (Cul3) pathway but despite this, cells exhibit a basal activation of Nrf2 target genes. It is yet not clear how Nrf2 maintains the expression of its targets under homeostatic conditions. Here, we found a stable 105 kDa Nrf2 form that is resistant to Keap1-Cul3-mediated degradation and translocates to the nucleus of lung cancer cells. RNA-Seq analysis indicate that it might originate from the exon 2 or exon 3-truncated transcripts. This stable 105 kDa Nrf2 form might help explain the constitutive activity of Nrf2 under normal cellular conditions.