Personalizing non-small cell lung cancer treatment through patient-derived xenograft models: preclinical and clinical factors for consideration.

PURPOSE: In the pursuit of creating personalized and more effective treatment strategies for lung cancer patients, Patient-Derived Xenografts (PDXs) have been introduced as preclinical platforms that can recapitulate the specific patient's tumor in an in vivo model. We investigated how well PDX models can preserve the tumor's clinical and molecular characteristics across different generations. METHODS: A Non-Small Cell Lung Cancer (NSCLC) PDX model was established in NSG-SGM3 mice and clinical and preclinical factors were assessed throughout subsequent passages. Our cohort consisted of 40 NSCLC patients, which were used to create 20 patient-specific PDX models in NSG-SGM3 mice. Histopathological staining and Whole Exome Sequencing (WES) analysis were preformed to understand tumor heterogeneity throughout serial passages. RESULTS: The main factors that contributed to the growth of the engrafted PDX in mice were a higher grade or stage of disease, in contrast to the long duration of chemotherapy treatment, which was negatively correlated with PDX propagation. Successful PDX growth was also linked to poorer prognosis and overall survival, while growth pattern variability was affected by the tumor aggressiveness, primarily affecting the first passage. Pathology analysis showed preservation of the histological type and grade; however, WES analysis revealed genomic instability in advanced passages, leading to the inconsistencies in clinically relevant alterations between the PDXs and biopsies. CONCLUSIONS: Our study highlights the impact of multiple clinical and preclinical factors on the engraftment success, growth kinetics, and tumor stability of patient-specific NSCLC PDXs, and underscores the importance of considering these factors when guiding and evaluating prolonged personalized treatment studies for NSCLC patients in these models, as well as signaling the imperative for additional investigations to determine the full clinical potential of this technique.

Digestive exophagy of biofilms by intestinal amoeba and its impact on stress tolerance and cytotoxicity.

The human protozoan parasite Entamoeba histolytica is responsible for amebiasis, a disease endemic to developing countries. E. histolytica trophozoites colonize the large intestine, primarily feeding on bacteria. However, in the gastrointestinal tract, bacterial cells form aggregates or structured communities called biofilms too large for phagocytosis. Remarkably, trophozoites are still able to invade and degrade established biofilms, utilizing a mechanism that mimics digestive exophagy. Digestive exophagy refers to the secretion of digestive enzymes that promote the digestion of objects too large for direct phagocytosis by phagocytes. E. histolytica cysteine proteinases (CPs) play a crucial role in the degradation process of Bacillus subtilis biofilm. These proteinases target TasA, a major component of the B. subtilis biofilm matrix, also contributing to the adhesion of the parasite to the biofilm. In addition, they are also involved in the degradation of biofilms formed by Gram-negative and Gram-positive enteric pathogens. Furthermore, biofilms also play an important role in protecting trophozoites against oxidative stress. This specific mechanism suggests that the amoeba has adapted to prey on biofilms, potentially serving as an untapped reservoir for novel therapeutic approaches to treat biofilms. Consistently, products derived from the amoeba have been shown to restore antibiotic sensitivity to biofilm cells. In addition, our findings reveal that probiotic biofilms can act as a protective shield for mammalian cells, hindering the progression of the parasite towards them.

Nonsense-mediated mRNA decay and cystic fibrosis.

Approximately one-third of the alleles causing genetic diseases carry premature termination codons (PTCs). Therapeutic approaches for mutations generating in-frame PTCs are aimed at promoting translational readthrough of the PTC, to enable the synthesis and expression of full-length functional proteins. Interestingly, readthrough studies in tissue culture cells, mouse models, and clinical trials revealed a wide variability in the response to the readthrough treatments. The molecular basis for this variability includes the identity of the PTC and its sequence context, the chemical composition of the readthrough drug, and, as we showed recently, the level of PTC-bearing transcripts. One post-transcriptional mechanism that specifically regulates the level of PTC-bearing transcripts is nonsense-mediated mRNA decay (NMD). We have previously shown a role for NMD in regulating the response of CF patients carrying CFTR PTCs to readthrough treatment. Here we describe all the protocols for analyzing CFTR nonsense transcript levels and for investigating the role of NMD in the response to readthrough treatment. This includes inhibition of the NMD mechanism, quantification of CFTR nonsense transcripts and physiologic NMD substrates, and analysis of the CFTR function.

Introducing sense into nonsense in treatments of human genetic diseases.

Approximately one-third of alleles causing genetic diseases carry premature termination codons (PTCs), which lead to the production of truncated proteins. The past decade has seen considerable interest in therapeutic approaches aimed at readthrough of in-frame PTCs to enable synthesis of full-length proteins. However, attempts to readthrough PTCs in many diseases resulted in variable effects. Here, we focus on the efforts of such therapeutic approaches in cystic fibrosis and Duchenne muscular dystrophy and discuss the factors contributing to successful readthrough and how the nonsense-mediated mRNA decay (NMD) pathway regulates this response. A deeper understanding of the molecular basis for variable response to readthrough of PTCs is necessary so that appropriate therapies can be developed to treat many human genetic diseases caused by PTCs.

The efficiency of nonsense-mediated mRNA decay is an inherent character and varies among different cells.

Nonsense-mediated mRNA decay (NMD) is a mechanism, which selectively degrades transcripts carrying premature termination codons (PTCs) and a variety of physiologic transcripts containing NMD-inducing features. In a recent study, we have found variable NMD efficiency among nasal epithelial cells obtained from cystic fibrosis (CF) patients. This variability was found for CF transmembrane conductance regulator (CFTR) transcripts carrying the W1282X PTC, as well as for several NMD physiologic substrates. Here, we aimed to investigate the possibility that variability in NMD efficiency is a more generalized phenomenon and is not restricted to nasal epithelial cells. To investigate this possibility, we analyzed the NMD efficiency of both a CFTR constructs carrying the W1282X PTC and beta-globin constructs carrying the NS39 PTC, in HeLa and MCF7 cells. Variability in NMD efficiency was found for both constructs between the cells, such that in HeLa cells the NMD was highly efficient and in MCF7 the efficiency was significantly lower. Moreover, similar differences in the efficiency of NMD were found for five endogenous NMD physiologic transcripts. Altogether, our results demonstrate existence of cells in which NMD of all transcripts is efficient, whereas others in which the NMD is less efficient, suggesting that the efficiency of NMD is an inherent character of cells. Our results also suggest that variability in the efficiency of NMD is a general phenomenon and is not restricted to nasal epithelial cells. As NMD affects the level of many transcripts, variability in the NMD efficiency might play a role as a genetic modifier of different cellular functions.

Nonsense-mediated mRNA decay affects nonsense transcript levels and governs response of cystic fibrosis patients to gentamicin.

Aminoglycosides can readthrough premature termination codons (PTCs), permitting translation of full-length proteins. Previously we have found variable efficiency of readthrough in response to the aminoglycoside gentamicin among cystic fibrosis (CF) patients, all carrying the W1282X nonsense mutation. Here we demonstrate that there are patients in whom the level of CF transmembrane conductance regulator (CFTR) nonsense transcripts is markedly reduced, while in others it is significantly higher. Response to gentamicin was found only in patients with the higher level. We further investigated the possibility that the nonsense-mediated mRNA decay (NMD) might vary among cells and hence governs the level of nonsense transcripts available for readthrough. Our results demonstrate differences in NMD efficiency of CFTR transcripts carrying the W1282X mutation among different epithelial cell lines derived from the same tissue. Variability was also found for 5 physiologic NMD substrates, RPL3, SC35 1.6 kb, SC35 1.7 kb, ASNS, and CARS. Importantly, our results demonstrate the existence of cells in which NMD of all transcripts was efficient and others in which the NMD was less efficient. Downregulation of NMD in cells carrying the W1282X mutation increased the level of CFTR nonsense transcripts and enhanced the CFTR chloride channel activity in response to gentamicin. Together our results suggest that the efficiency of NMD might vary and hence have an important role in governing the response to treatments aiming to promote readthrough of PTCs in many genetic diseases.