In vitro functional assays to assess the reciprocal interplay between tumor cells and macrophages.

Tumor-associated macrophages (TAMs) are integral components of the tumor microenvironment. They are involved in various aspects of tumor cell biology, driving pathological processes such as tumor cell proliferation, metastasis, immunosuppression, and resistance to therapy. TAMs exert their tumorigenic effects by secreting growth factors, cytokines/chemokines, metabolites, and other soluble bioactive molecules. These mediators directly promote tumor cell proliferation and modulate interactions with immune and stromal cells, facilitating further tumor growth. As research into therapies targeting TAMs intensifies, there is a growing need for reliable methods to comprehend the impact of TAMs on cancer progression and to validate novel therapeutics directed at TAMs. The traditional "M1-M2" macrophage classification based on transcriptional profiles of TAMs is not only too simplistic to describe their physiological roles, it also does not explain differences observed between mouse and human macrophages. In this context, methods that assess how TAMs influence tumor or immune cells, either through direct contact or the release of soluble factors, offer a more promising approach. We describe here comprehensive protocols for in vitro functional assays to study TAMs, specifically regarding their impact on the growth of lung cancer cells. We have applied these methods to both mouse and human macrophages, achieving similar outcomes in promoting the proliferation of cancer cells. This methodology can serve as a standardized approach for testing novel therapeutic approaches, targeting TAMs with novel immunotherapeutic compounds, or utilizing gene-editing techniques. Taken together, the described methodology may contribute to our understanding of complex macrophage-tumor interactions and support the development of innovative therapeutic strategies.

Pericentromeric satellite lncRNAs are induced in cancer-associated fibroblasts and regulate their functions in lung tumorigenesis.

The abnormal tumor microenvironment (TME) often dictates the therapeutic response of cancer to chemo- and immuno-therapy. Aberrant expression of pericentromeric satellite repeats has been reported for epithelial cancers, including lung cancer. However, the transcription of tandemly repetitive elements in stromal cells of the TME has been unappreciated, limiting the optimal use of satellite transcripts as biomarkers or anti-cancer targets. We found that transcription of pericentromeric satellite DNA (satDNA) in mouse and human lung adenocarcinoma was observed in cancer-associated fibroblasts (CAFs). In vivo, lung fibroblasts expressed pericentromeric satellite repeats HS2/HS3 specifically in tumors. In vitro, transcription of satDNA was induced in lung fibroblasts in response to TGFß, IL1α, matrix stiffness, direct contact with tumor cells and treatment with chemotherapeutic drugs. Single-cell transcriptome analysis of human lung adenocarcinoma confirmed that CAFs were the cell type with the highest number of satellite transcripts. Human HS2/HS3 pericentromeric transcripts were detected in the nucleus, cytoplasm, extracellularly and co-localized with extracellular vesicles in situ in human biopsies and activated fibroblasts in vitro. The transcripts were transmitted into recipient cells and entered their nuclei. Knock-down of satellite transcripts in human lung fibroblasts attenuated cellular senescence and blocked the formation of an inflammatory CAFs phenotype which resulted in the inhibition of their pro-tumorigenic functions. In sum, our data suggest that satellite long non-coding (lnc) RNAs are induced in CAFs, regulate expression of inflammatory genes and can be secreted from the cells, which potentially might present a new element of cell-cell communication in the TME.

EZH2 Inhibition as New Epigenetic Treatment Option for Pancreatic Neuroendocrine Neoplasms (PanNENs).

Pancreatic neuroendocrine neoplasms are epigenetically driven tumors, but therapies against underlying epigenetic drivers are currently not available in the clinical practice. We aimed to investigate EZH2 (Enhancer of Zest homolog) expression in PanNEN and the impact of EZH2 inhibition in three different PanNEN preclinical models. EZH2 expression in PanNEN patient samples (n = 172) was assessed by immunohistochemistry and correlated with clinico-pathological data. Viability of PanNEN cell lines treated with EZH2 inhibitor (GSK126) was determined in vitro. Lentiviral transduction of shRNA targeting EZH2 was performed in QGP1 cells, and cell proliferation was measured. Rip1TAG2 mice underwent GSK126 treatment for three weeks starting from week 10 of age. Primary cells isolated from PanNEN patients (n = 6) were cultivated in 3D as islet-like tumoroids and monitored for 10 consecutive days upon GSK126 treatment. Viability was measured continuously for the whole duration of the treatment. We found that high EZH2 expression correlated with higher tumor grade (p < 0.001), presence of distant metastases (p < 0.001), and shorter disease-free survival (p < 0.001) in PanNEN patients. Inhibition of EZH2 in vitro in PanNEN cell lines and in patient-derived islet-like tumoroids reduced cell viability and impaired cell proliferation, while inhibition of EZH2 in vivo in Rip1TAG2 mice reduced tumor burden. Our results show that EZH2 is highly expressed in high-grade PanNENs, and during disease progression it may contribute to aberrations in the epigenetic cellular landscape. Targeting EZH2 may represent a valuable epigenetic treatment option for patients with PanNEN.

Biodegradable Polymeric Multilayer Capsules for Therapy of Lung Cancer.

Formulated forms of cancer therapeutics enhance the efficacy of treatment by more precise targeting, increased bioavailability of drugs, and an aptitude of some delivery systems to overcome multiple drug resistance of tumors. Drug carriers acquire importance for anti-cancer interventions via targeting tumor-associated macrophages with active molecules capable to either eliminate them or change their polarity. Although several packaged drug forms have reached the market, there is still a high demand for novel carrier systems to hurdle limitations of existing drugs on active molecules, toxicity, bioeffect, and stability. Here, we report a facile assembly and delivery methodology for biodegradable polymeric multilayer capsules (PMC) with the purpose of further use in injectable drug formulations for lung cancer therapy via direct erosion of tumors and suppression of the tumor-promoting function of macrophages in the tumor microenvironment. We demonstrate delivery of low-molecular-weight drug molecules to lung cancer cells and macrophages and provide details on in vivo distribution, cellular uptake, and disintegration of the developed PMC. Poly-l-arginine and dextran sulfate alternately adsorb on a ∼500 nm CaCO3 sacrificial template followed by removal of the inorganic core to obtain hollow capsules for consequent loading with drug molecules, gemcitabine or clodronate. The capsules further compacted upon loading down to ∼250 nm in diameter via heat treatment. A comparative study of the capsule internalization rate in vitro and in vivo reveals the benefits of a diminished carrier size. We show that macrophages and epithelial cells of the lungs and liver internalize capsules with efficacy higher than 75%. Using an in vivo mouse model of lung cancer, we also confirm that tumor lungs better retain smaller capsules than the healthy lung tissue. The pronounced cytotoxic effect of the encapsulated gemcitabine on lung cancer cells and the ability of the encapsulated clodronate to block the tumor-promoting function of macrophages prove the efficacy of the developed capsule loading method in vitro. Our study taken as a whole demonstrates the great potential of the developed PMC for in vivo treatment of cancer via transporting active molecules, including those that are water-soluble with low molecular weight, to both cancer cells and macrophages through the bloodstream.

Rosmarinus officinalis L. extract ameliorates intestinal inflammation through MAPKs/NF-κB signaling in a murine model of acute experimental colitis.

We investigated the anti-inflammatory and anti-colitis effects of Rosmarinus officinalis L. extract (RE) by using both in vitro LPS-activated mouse RAW 264.7 macrophages and in vivo dextran sulfate sodium (DSS)-induced experimental murine colitis and suggested the underlying possible mechanisms. Liquid Chromatography-Mass Spectrometry (LC-MS) analysis was performed to identify the major components present in the RE. The clinical signs, biochemistry, immunoblot, ELISA and histology in colon tissues were assessed in order to elucidate the beneficial effect of RE. RE suppressed the LPS-induced pro-inflammatory cytokine production and the expressions of inflammatory proteins in macrophages. Administration of RE (50 and 100 mg kg(-1)) also significantly reduced the severity of DSS-induced murine colitis, as assessed by the clinical symptoms, colon length and histology. RE administration prevented the DSS-induced activation of p38, ERK and JNK MAPKs, attenuated IκBα phosphorylation and subsequent nuclear translocation and DNA binding of NF-κB (p65). RE also suppressed the COX-2 and iNOS expressions, decreased the levels of TNF-α and IL-6 cytokines and the myeloperoxidase activity in the colon tissue. Histological observation revealed that RE administration alleviated mucosal damage and inflammatory cell infiltration induced by DSS in the colon tissue. Hence, RE could be used as a new preventive and therapeutic food ingredient or as a dietary supplement for inflammatory bowel disease.