Stromal cells engineered to express T cell factors induce robust CLL cell proliferation in vitro and in PDX co-transplantations allowing the identification of RAF inhibitors as anti-proliferative drugs.

Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.

In-cell NMR suggests that DNA i-motif levels are strongly depleted in living human cells.

I-Motifs (iM) are non-canonical DNA structures potentially forming in the accessible, single-stranded, cytosine-rich genomic regions with regulatory roles. Chromatin, protein interactions, and intracellular properties seem to govern iM formation at sites with i-motif formation propensity (iMFPS) in human cells, yet their specific contributions remain unclear. Using in-cell NMR with oligonucleotide iMFPS models, we monitor iM-associated structural equilibria in asynchronous and cell cycle-synchronized HeLa cells at 37 °C. Our findings show that iMFPS displaying pHT < 7 under reference in vitro conditions occur predominantly in unfolded states in cells, while those with pHT > 7 appear as a mix of folded and unfolded states depending on the cell cycle phase. Comparing these results with previous data obtained using an iM-specific antibody (iMab) reveals that cell cycle-dependent iM formation has a dual origin, and iM formation concerns only a tiny fraction (possibly 1%) of genomic sites with iM formation propensity. We propose a comprehensive model aligning observations from iMab and in-cell NMR and enabling the identification of iMFPS capable of adopting iM structures under physiological conditions in living human cells. Our results suggest that many iMFPS may have biological roles linked to their unfolded states.

Plastic response of macrophages to metal ions and nanoparticles in time mimicking metal implant body environment.

The paradigm of using metal biomaterials could be viewed from two sides - treatment of wide spectrum of degenerative diseases, and debris release from materials. After implant insertion, metal nanoparticles (NPs) and ions are released not only upon the first contact with cells/tissues, but in continual manner, which is immediately recognized by immune cells. In this work, the effects of metal nanoparticles (TiO2, Ni) and ions (Ni2+, Co2+, Cr3+, Mo6+) on primary human M0 macrophages from the blood samples of osteoarthritic patients undergoing total arthroplasty were studied in order to monitor immunomodulatory effects on the cells in a real-time format. The highest NiNPs concentration of 10 µg/ml had no effect on any of macrophage parameters, while the Ni2+ ions cytotoxicity limit for the cells is 0.5 mM. The cytotoxic effects of higher Ni2+ concentration revealed mitochondrial network fragmentation leading to mitochondrial dysfunction, accompanied by increased lysosomal activity and changes in pro-apoptotic markers. The suppression of M2 cell formation ability was connected to presence of Ni2+ ions (0.5 mM) and TiO2NPs (10 µg/ml). The immunomodulatory effect of Mo6+ ions, controversially, inhibit the formation of the cells with M1 phenotype and potentiate the thread-like shape M2s with increased chaotic cell movement. To summarize, metal toxicity depends on the debris form. Both, metal ions and nanoparticles affect macrophage size, morphological and functional parameters, but the effect of ions is more complex and likely more harmful, which has potential impact on healing and determines post-implantation reactions.