Tunable PhenoCycler imaging of the murine pre-clinical tumour microenvironments.

BACKGROUND: The tumour microenvironment (TME) consists of tumour-supportive immune cells, endothelial cells, and fibroblasts. PhenoCycler, a high-plex single cell spatial biology imaging platform, is used to characterize the complexity of the TME. Researchers worldwide harvest and bank tissues from mouse models which are employed to model a plethora of human disease. With the explosion of interest in spatial biology, these panoplies of archival tissues provide a valuable resource to answer new questions. Here, we describe our protocols for developing tunable PhenoCycler multiplexed imaging panels and describe our open-source data analysis pipeline. Using these protocols, we used PhenoCycler to spatially resolve the TME of 8 routinely employed pre-clinical models of lymphoma, breast cancer, and melanoma preserved as FFPE. RESULTS: Our data reveal distinct TMEs in the different cancer models that were imaged and show that cell-cell contacts differ depending on the tumour type examined. For instance, we found that the immune infiltration in a murine model of melanoma is altered in cellular organization in melanomas that become resistant to αPD-1 therapy, with depletions in a number of cell-cell interactions. CONCLUSIONS: This work presents a valuable resource study seamlessly adaptable to any field of research involving murine models. The methodology described allows researchers to address newly formed hypotheses using archival materials, bypassing the new to perform new mouse studies.

STAT6 mutations enriched at diffuse large B-cell lymphoma relapse reshape the tumor microenvironment.

Diffuse large B-cell lymphoma (DLBCL) relapses in approximately 40% of patients following frontline therapy. We reported that STAT6D419 mutations are enriched in relapsed/refractory DLBCL (rrDLBCL) samples, suggesting that JAK/STAT signaling plays a role in therapeutic resistance. We hypothesized that STAT6D419 mutations can improve DLBCL cell survival by reprogramming the microenvironment to sustain STAT6 activation. Thus, we investigated the role of STAT6D419 mutations on DLBCL cell growth and its microenvironment. We found that phospho-STAT6D419N was retained in the nucleus longer than phospho-STAT6WT following IL-4 stimulation, and STAT6D419N recognized a more restricted DNA-consensus sequence than STAT6WT. Upon IL-4 induction, STAT6D419N expression led to a higher magnitude of gene expression changes, but in a more selective list of gene targets compared with STATWT. The most significantly expressed genes induced by STAT6D419N were those implicated in survival, proliferation, migration, and chemotaxis, in particular CCL17. This chemokine, also known as TARC, attracts helper T-cells to the tumor microenvironment, especially in Hodgkin's lymphoma. To this end, in DLBCL, phospho-STAT6+ rrDLBCL cells had a greater proportion of infiltrating CD4+ T-cells than phospho-STAT6- tumors. Our findings suggest that STAT6D419 mutations in DLBCL lead to cell autonomous changes, enhanced signaling, and altered composition of the tumor microenvironment.

The role of eIF4F-driven mRNA translation in regulating the tumour microenvironment.

Cells can rapidly adjust their proteomes in dynamic environments by regulating mRNA translation. There is mounting evidence that dysregulation of mRNA translation supports the survival and adaptation of cancer cells, which has stimulated clinical interest in targeting elements of the translation machinery and, in particular, components of the eukaryotic initiation factor 4F (eIF4F) complex such as eIF4E. However, the effect of targeting mRNA translation on infiltrating immune cells and stromal cells in the tumour microenvironment (TME) has, until recently, remained unexplored. In this Perspective article, we discuss how eIF4F-sensitive mRNA translation controls the phenotypes of key non-transformed cells in the TME, with an emphasis on the underlying therapeutic implications of targeting eIF4F in cancer. As eIF4F-targeting agents are in clinical trials, we propose that a broader understanding of their effect on gene expression in the TME will reveal unappreciated therapeutic vulnerabilities that could be used to improve the efficacy of existing cancer therapies.

The sigma-1 receptor behaves as an atypical auxiliary subunit to modulate the functional characteristics of Kv1.2 channels expressed in HEK293 cells.

Expression of Kv1.2 within Kv1.x potassium channel complexes is critical in maintaining appropriate neuronal excitability and determining the threshold for action potential firing. This is attributed to the interaction of Kv1.2 with a hitherto unidentified protein that confers bimodal channel activation gating, allowing neurons to adapt to repetitive trains of stimulation and protecting against hyperexcitability. One potential protein candidate is the sigma-1 receptor (Sig-1R), which regulates other members of the Kv1.x channel family; however, the biophysical nature of the interaction between Sig-1R and Kv1.2 has not been elucidated. We hypothesized that Sig-1R may regulate Kv1.2 and may further act as the unidentified modulator of Kv1.2 activation. In transiently transfected HEK293 cells, we found that ligand activation of the Sig-1R modulates Kv1.2 current amplitude. More importantly, Sig-1R interacts with Kv1.2 in baseline conditions to influence bimodal activation gating. These effects are abolished in the presence of the auxiliary subunit Kvß2 and when the Sig-1R mutation underlying ALS16 (Sig-1R-E102Q), is expressed. These data suggest that Kvß2 occludes the interaction of Sig-1R with Kv1.2, and that E102 may be a residue critical for Sig-1R modulation of Kv1.2. The results of this investigation describe an important new role for Sig-1R in the regulation of neuronal excitability and introduce a novel mechanism of pathophysiology in Sig-1R dysfunction.