Impact of soluble tumor necrosis factor-related apoptosis-inducing ligand released by engineered adipose mesenchymal stromal cells on white blood cells.

BACKGROUND AIMS: The proapoptotic protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is physiologically expressed by immune cells and performs regulatory functions in infections, autoimmune diseases and cancer, where it acts as a tumor suppressor. Adipose-derived mesenchymal stromal cells (AD-MSCs) also may play immunomodulatory roles in both primary and acquired immune responses. We have previously demonstrated the efficacy of an anticancer gene therapy based on AD-MSC engineered to secrete a soluble TRAIL variant (sTRAIL) against pancreatic cancer. However, the impact of AD-MSC sTRAIL on leukocyte subsets has been not yet considered also to predict a possible immunotoxicity profile in the clinical translation of this cell-based anticancer strategy. METHODS: Monocytes, polymorphonuclear cells and T lymphocytes were freshly isolated from the peripheral blood of healthy donors. Immunophenotype and functional (DR4 and DR5) and decoy (DcR1 and DcR2) TRAIL receptors were tested by flow cytometry. The viability of white blood cells treated with sTRAIL released by gene-modified AD-MSC or co-cultured with AD-MSC sTRAIL was then evaluated by both metabolic assays and flow cytometry. In addition, cytokine profile in co-cultures was analyzed by multiplex enzyme-linked immunosorbent assay. RESULTS: Monocytes and polymorphonuclear cells showed high positivity for DR5 and DcR2, respectively, whereas T cells revealed negligible expression of all TRAIL receptors. Irrespective of TRAIL receptors' presence on the cell membrane, white blood cells were refractory to the proapoptotic effect displayed by sTRAIL secreted by gene-modified AD-MSC, and direct cell-to-cell contact with AD-MSC sTRAIL had negligible impact on T-cell and monocyte viability. Cytokine crosstalk involving interleukin 10, tumor necrosis factor alpha, and interferon gamma secreted by T lymphocytes and vascular endothelial growth factor A and interleukin 6 released by AD-MSC was highlighted in T-cell and AD-MSC sTRAIL co-cultures. CONCLUSIONS: In summary, this study demonstrates the immunological safety and thus the clinical feasibility of an anticancer approach based on AD-MSC expressing the proapoptotic molecule sTRAIL.

Deepening the Knowledge of ROS1 Rearrangements in Non-Small Cell Lung Cancer: Diagnosis, Treatment, Resistance and Concomitant Alterations.

ROS proto-oncogene 1 (ROS1) rearrangements are reported in about 1-2% of non-squamous non-small-cell lung cancer (NSCLC). After efficacy of crizotinib was demonstrated, identification of ROS1 translocations in advanced disease became fundamental to give patients the chance of specific and effective treatment. Different methods are available for detection of rearrangements, and probably the real prevalence of ROS1 rearrangements is higher than that reported in literature, as our capacity to detect gene rearrangements is improving. In particular, with next generation sequencing (NGS) techniques, we are currently able to assess multiple genes simultaneously with increasing sensitivity. This is leading to overcome the "single oncogenic driver" paradigm, and in the very near future, the co-existence of multiple drivers will probably emerge more frequently and represent a therapeutic issue. Since recently, crizotinib has been the only available therapy, but today, many other tyrosine kinase inhibitors (TKI) are emerging and seem promising both in first and subsequent lines of treatment. Indeed, novel inhibitors are also able to overcome resistance mutations to crizotinib, hypothesizing a possible sequential strategy also in ROS1-rearranged disease. In this review, we will focus on ROS1 rearrangements, dealing with diagnostic aspects, new therapeutic options, resistance issues and the coexistence of ROS1 translocations with other molecular alterations.

In vitro and in vivo discrepancy in inducing apoptosis by mesenchymal stromal cells delivering membrane-bound tumor necrosis factor-related apoptosis inducing ligand in osteosarcoma pre-clinical models.

BACKGROUND: Osteosarcoma (OS) is the most frequent pediatric malignant bone tumor. OS patients have not seen any major therapeutic progress in the last 30 years, in particular in the case of metastatic disease, which requires new therapeutic strategies. The pro-apoptotic cytokine Tumor necrosis factor (TNF)-Related Apoptosis Inducing Ligand (TRAIL) can selectively kill tumor cells while sparing normal cells, making it a promising therapeutic tool in several types of cancer. However, many OS cell lines appear resistant to recombinant human (rh)TRAIL-induced apoptosis. We, therefore, hypothesized that TRAIL presentation at the membrane level of carrier cells might overcome this resistance and trigger apoptosis. METHODS: To address this, human adipose mesenchymal stromal cells (MSCs) transfected in a stable manner to express membrane-bound full-length human TRAIL (mbTRAIL) were co-cultured with several human OS cell lines. RESULTS: This induced apoptosis by cell-to-cell contact even in cell lines initially resistant to rhTRAIL. In contrast, mbTRAIL delivered by MSCs was not able to counteract tumor progression in this OS orthotopic model. DISCUSSION: This was partly due to the fact that MSCs showed a potential to support tumor development. Moreover, the expression of mbTRAIL did not show caspase activation in adjacent tumor cells.

TRAIL delivered by mesenchymal stromal/stem cells counteracts tumor development in orthotopic Ewing sarcoma models.

Ewing sarcoma (EWS) is the second most frequent pediatric malignant bone tumor. EWS patients have not seen any major therapeutic progress in the last 30 years, in particular in the case of metastatic disease, which requires new therapeutic strategies. The pro-apoptotic cytokine TNF-Related Apoptosis Inducing Ligand (TRAIL) can selectively kill tumor cells while sparing normal cells, making it a promising therapeutic tool in several types of cancer. However, certain EWS cell lines appear resistant to recombinant human (rh) TRAIL-induced apoptosis. We therefore hypothesized that a TRAIL presentation at the surface of the carrier cells might overcome this resistance and trigger apoptosis. For this purpose, human adipose mesenchymal stromal/stem cells (MSC) transfected in a stable manner to express full-length human TRAIL were co-cultured with several human EWS cell lines, inducing apoptosis by cell-to-cell contact even in cell lines initially resistant to rhTRAIL or AMG655, an antibody agonist to the death receptor, DR5. In vivo, TRAIL delivered by MSCs was able to counteract tumor progression in two orthotopic models of Ewing sarcoma, associated with caspase activation, indicating that a cell-based delivery of a potent apoptosis-inducing factor could be relevant in EWS.

Mesenchymal progenitors aging highlights a miR-196 switch targeting HOXB7 as master regulator of proliferation and osteogenesis.

Human aging is associated with a decrease in tissue functions combined with a decline in stem cells frequency and activity followed by a loss of regenerative capacity. The molecular mechanisms behind this senescence remain largely obscure, precluding targeted approaches to counteract aging. Focusing on mesenchymal stromal/stem cells (MSC) as known adult progenitors, we identified a specific switch in miRNA expression during aging, revealing a miR-196a upregulation which was inversely correlated with MSC proliferation through HOXB7 targeting. A forced HOXB7 expression was associated with an improved cell growth, a reduction of senescence, and an improved osteogenesis linked to a dramatic increase of autocrine basic fibroblast growth factor secretion. These findings, along with the progressive decrease of HOXB7 levels observed during skeletal aging in mice, indicate HOXB7 as a master factor driving progenitors behavior lifetime, providing a better understanding of bone senescence and leading to an optimization of MSC performance.

Mesenchymal progenitors expressing TRAIL induce apoptosis in sarcomas.

Sarcomas are frequent tumors in children and young adults that, despite a relative chemo-sensitivity, show high relapse rates with up to 80% of metastatic patients dying in 5 years from diagnosis. The real ontogeny of sarcomas is still debated and evidences suggest they may derive from precursors identified within mesenchymal stromal/stem cells (MSC) fractions. Recent studies on sarcoma microenvironment additionally indicated that MSC could take active part in generation of a supportive stroma. Based on this knowledge, we conceived to use modified MSC to deliver tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) targeting different sarcoma histotypes. Gene modified MSC expressing TRAIL were cocultured with different osteosarcoma, rhabdomyosarcoma, and Ewing's Sarcoma (ES) cell lines assessing viability and caspase-8 activation. An in vivo model focused on ES was then implemented considering the impact of MSC-TRAIL on tumor size, apoptosis, and angiogenesis. MSC expressing TRAIL induced significantly high apoptosis in all tested lines. Sarcoma death was specifically associated with caspase-8 activation starting from 8 hours of coculture with MSC-TRAIL. When injected into pre-established ES xenotransplants, MSC-TRAIL persisted within its stroma, causing significant tumor apoptosis versus control groups. Additional histological and in vitro studies reveal that MSC-TRAIL could also exert potent antiangiogenic functions. Our results suggest that MSC as TRAIL vehicles could open novel therapeutic opportunities for sarcoma by multiple mechanisms.

Mesenchymal stem/stromal cells as a delivery platform in cell and gene therapies.

Regenerative medicine relying on cell and gene therapies is one of the most promising approaches to repair tissues. Multipotent mesenchymal stem/stromal cells (MSC), a population of progenitors committing into mesoderm lineages, are progressively demonstrating therapeutic capabilities far beyond their differentiation capacities. The mechanisms by which MSC exert these actions include the release of biomolecules with anti-inflammatory, immunomodulating, anti-fibrogenic, and trophic functions. While we expect the spectra of these molecules with a therapeutic profile to progressively expand, several human pathological conditions have begun to benefit from these biomolecule-delivering properties. In addition, MSC have also been proposed to vehicle genes capable of further empowering these functions. This review deals with the therapeutic properties of MSC, focusing on their ability to secrete naturally produced or gene-induced factors that can be used in the treatment of kidney, lung, heart, liver, pancreas, nervous system, and skeletal diseases. We specifically focus on the different modalities by which MSC can exert these functions. We aim to provide an updated understanding of these paracrine mechanisms as a prerequisite to broadening the therapeutic potential and clinical impact of MSC.

IGF-1-mediated osteoblastic niche expansion enhances long-term hematopoietic stem cell engraftment after murine bone marrow transplantation.

The efficiency of hematopoietic stem cell (HSC) engraftment after bone marrow (BM) transplantation depends largely on the capacity of the marrow microenvironment to accept the transplanted cells. While radioablation of BM damages osteoblastic stem cell niches, little is known about their restoration and mechanisms governing their receptivity to engraft transplanted HSCs. We previously reported rapid restoration and profound expansion of the marrow endosteal microenvironment in response to marrow radioablation. Here, we show that this reorganization represents proliferation of mature endosteal osteoblasts which seem to arise from a small subset of high-proliferative, relatively radio-resistant endosteal cells. Multiple layers of osteoblasts form along the endosteal surface within 48 hours after total body irradiation, concomitant with a peak in marrow cytokine expression. This niche reorganization fosters homing of the transplanted hematopoietic cells to the host marrow space and engraftment of long-term-HSC. Inhibition of insulin-like growth factor (IGF)-1-receptor tyrosine kinase signaling abrogates endosteal osteoblast proliferation and donor HSC engraftment, suggesting that the cytokine IGF-1 is a crucial mediator of endosteal niche reorganization and consequently donor HSC engraftment. Further understanding of this novel mechanism of IGF-1-dependent osteoblastic niche expansion and HSC engraftment may yield clinical applications for improving engraftment efficiency after clinical HSC transplantation.

IFN-ß expression is directly activated in human neutrophils transfected with plasmid DNA and is further increased via TLR-4-mediated signaling.

Upon LPS binding, TLR4 activates a MyD88-dependent pathway leading to the transcriptional activation of proinflammatory genes, as well as a MyD88-independent/TRIF-dependent pathway, responsible for the transcriptional induction of IFN-ß. Previous findings delineated that human neutrophils are unable to induce the transcription of IFN-ß in response to TLR4 stimulation. Because neutrophils do not express protein kinase C ε, a molecule recently reported as essential for initiating the MyD88-independent/TRIF-dependent pathway, we optimized an electroporation method to transfect PKCε into neutrophils with very high efficiency. By doing so, a significant IFN-ß mRNA expression was induced, in the absence of LPS stimulation, not only in PKCε-overexpressing neutrophils but also in cells transfected with a series of empty DNA plasmids; however, LPS further upregulated the IFN-ß transcript levels in plasmid-transfected neutrophils, regardless of PKCε overexpression. Phosphoimmunoblotting studies, as well as chromatin immunoprecipitation assays targeting the IFN-ß promoter, revealed that IFN-ß mRNA induction occurred through the cooperative action of IRF3, activated by transfected DNA, and NF-κB, activated by LPS. Additional immunoblotting and coimmunoprecipitation studies revealed that neutrophils constitutively express various cytosolic DNA sensors, including IFN-inducible protein 16, leucine-rich repeat (in Flightless I) interacting protein-1, and DDX41, as well as that IFN-inducible protein 16 is the intracellular receptor recognizing transfected DNA. Consistently, infection of neutrophils with intracellular pathogens, such as Bartonella henselae, Listeria monocytogenes, Legionella pneumophila, or adenovirus type 5, promoted a marked induction of IFN-ß mRNA expression. Taken together, these data raise questions about the role of PKCε in driving the MyD88-independent/TRIF-dependent response and indicate that human neutrophils are able to recognize and respond to microbial cytosolic DNA.

Expression of HOXB7 in the Lung of Patients with Idiopathic Pulmonary Fibrosis: A Proof-of-Concept Study.

BACKGROUND: The molecular pathways involved in the onset and progression of idiopathic pulmonary fibrosis (IPF) still need to be fully clarified as some are shared with lung cancer development. HOXB7, a member of the homeobox (Hox) gene family, has been found involved in various cancers. METHODS: Immunohistochemical (IHC) analysis was run on lung tissue samples from surgical lung biopsy (SLB) of 19 patients with IPF, retrospectively selected from the IPF database of the University Hospital of Modena. HOXB7 expression was analyzed and compared with that of five patients with no evidence of pulmonary fibrosis as controls. RESULTS: The semi-quantitative analysis of IHC showed that HOXB7 protein expression was higher in IPF patients compared to controls (difference between means = 6.2 ± 2.37, p = 0.0157). Further, HOXB7 expression was higher in IPF patients with a higher extent of fibrosis (50-75%)-measured with high-resolution computer tomography-compared to those with a lower extent (0-25%) (difference between means = 25.74 ± 6.72, p = 0.004). CONCLUSIONS: The expression of HOXB7 is higher in the lung of IPF patients compared to controls, and was represented in different cellular compartments within the lung niche. Further investigations are needed to clarify its role in the pathogenesis and progression of IPF.

Autologous anti-GD2 CAR T cells efficiently target primary human glioblastoma.

Glioblastoma (GBM) remains a deadly tumor. Treatment with chemo-radiotherapy and corticosteroids is known to impair the functionality of lymphocytes, potentially compromising the development of autologous CAR T cell therapies. We here generated pre-clinical investigations of autologous anti-GD2 CAR T cells tested against 2D and 3D models of GBM primary cells. We detected a robust antitumor effect, highlighting the feasibility of developing an autologous anti-GD2 CAR T cell-based therapy for GBM patients.

Combining gemcitabine and MSC delivering soluble TRAIL to target pancreatic adenocarcinoma and its stroma.

Pancreatic ductal adenocarcinoma (PDAC) still has a poor response to therapies, partly due to their cancer-associated fibroblasts (CAFs). Here, we investigate the synergistic impact of a combinatory approach between a known chemotherapy agent, such as gemcitabine (GEM), and gene-modified human mesenchymal stromal/stem cells (MSCs) secreting the pro-apoptotic soluble (s)TRAIL (sTRAIL MSCs) on both PDAC cells and CAFs. The combo significantly impacts on PDAC survival in 2D and 3D models. In orthotopic xenograft models, GEM and sTRAIL MSCs induce tumor architecture shredding with a reduction of CK7- and CK8/18-positive cancer cells and the abrogation of spleen metastases. A cytotoxic effect on primary human CAFs is also observed along with an alteration of their transcriptome and a reduction of the related desmoplasia. Collectively, we demonstrate a promising therapeutic profile of combining GEM and sTRAIL MSCs to target both tumoral and stromal compartments in PDAC.

Adipose stromal/stem cells assist fat transplantation reducing necrosis and increasing graft performance.

Autologous fat transfer (AFT) is a procedure for adipose tissue (AT) repair after trauma, burns, post-tumor resections and lipodystrophies still negatively impacted by the lack of graft persistence. The reasons behind this poor outcome are unclear and seem to involve damages in either harvested/transplanted mature adipocytes or on their mesenchymal progenitors, namely adipose stromal/stem cells (ASC), and due to post-transplant AT apoptosis and involution. A rabbit subcutaneous AT regeneration model was here developed to first evaluate graft quality at different times after implant focusing on related parameters, such as necrosis and vasculogenesis. Standard AFT was compared with a strategy where purified autologous ASC, combined with hyaluronic acid (HA), assisted AFT. Five million of autologous ex vivo isolated CD29+, CD90+, CD49e+ ASC, loaded into HA, enriched 1 ml of AT generating an early significant protective effect in reducing AFT necrosis and increasing vasculogenesis with a preservation of transplanted AT architecture. This beneficial impact of ASC assisted AFT was then confirmed at three months with a robust lipopreservation and no signs of cellular transformation. By a novel ASC assisted AFT approach we ensure a reduction in early cell death favoring an enduring graft performance possibly for a more stable benefit in patients.

Chasing the Role of miRNAs in RCC: From Free-Circulating to Extracellular-Vesicle-Derived Biomarkers.

Renal cell carcinoma (RCC) is the second most common cancer of the urinary system. The current therapeutic strategies are based on partial or total nephrectomy and/or targeted therapies based on immune checkpoint inhibitors to which patients are often refractory. Preventive and screening strategies do not exist and the few available biomarkers for RCC are characterized by a lack of sensitivity, outlining the need for novel noninvasive and sensitive biomarkers for early diagnosis and better disease monitoring. Blood liquid biopsy (LB) is a non- or minimally invasive procedure for a more representative view of tumor heterogeneity than a tissue biopsy, potentially allowing the real-time monitoring of cancer evolution. Growing interest is focused on the extracellular vesicles (EVs) secreted by either healthy or tumoral cells and recovered in a variety of biological matrices, blood included. EVs are involved in cell-to-cell crosstalk transferring their mRNAs, microRNAs (miRNAs), and protein content. In particular, transferred miRNAs may regulate tumorigenesis and proliferation also impacting resistance to apoptosis, thus representing potential useful biomarkers. Here, we present the latest efforts in the identification of circulating miRNAs in blood samples, focusing on the potential use of EV-derived miRNAs as RCC diagnostic and prognostic markers.

Inhibition of ERK1/2 signaling prevents bone marrow fibrosis by reducing osteopontin plasma levels in a myelofibrosis mouse model.

Clonal myeloproliferation and development of bone marrow (BM) fibrosis are the major pathogenetic events in myelofibrosis (MF). The identification of novel antifibrotic strategies is of utmost importance since the effectiveness of current therapies in reverting BM fibrosis is debated. We previously demonstrated that osteopontin (OPN) has a profibrotic role in MF by promoting mesenchymal stromal cells proliferation and collagen production. Moreover, increased plasma OPN correlated with higher BM fibrosis grade and inferior overall survival in MF patients. To understand whether OPN is a druggable target in MF, we assessed putative inhibitors of OPN expression in vitro and identified ERK1/2 as a major regulator of OPN production. Increased OPN plasma levels were associated with BM fibrosis development in the Romiplostim-induced MF mouse model. Moreover, ERK1/2 inhibition led to a remarkable reduction of OPN production and BM fibrosis in Romiplostim-treated mice. Strikingly, the antifibrotic effect of ERK1/2 inhibition can be mainly ascribed to the reduced OPN production since it could be recapitulated through the administration of anti-OPN neutralizing antibody. Our results demonstrate that OPN is a novel druggable target in MF and pave the way to antifibrotic therapies based on the inhibition of ERK1/2-driven OPN production or the neutralization of OPN activity.

Phosphorylation of serine 21 modulates the proliferation inhibitory more than the differentiation inducing effects of C/EBPα in K562 cells.

The CCAAT/enhancer binding protein α (C/EBPα) is a transcription factor required for differentiation of myeloid progenitors. In acute myeloid leukemia (AML) cells expressing the constitutively active FLT3-ITD receptor tyrosine kinase, MAP kinase-dependent phosphorylation of serine 21 (S21) inhibits the ability of C/EBPα to induce granulocytic differentiation. To assess whether this post-translational modification also modulates the activity of C/EBPα in BCR/ABL-expressing cells, we tested the biological effects of wild-type and mutant C/EBPα mimicking phosphorylated or non-phosphorylatable serine 21 (S21D and S21A, respectively) in K562 cells ectopically expressing tamoxifen-regulated C/EBPα-ER chimeric proteins. We show here that S21D C/EBPα-ER induced terminal granulocytic differentiation of K562 cells almost as well as wild-type C/EBPα-ER, while S21A C/EBPα-ER was less efficient. Furthermore, wild-type C/EBPα suppressed the proliferation and colony formation of K562 cells vigorously, while S21D and S21A C/EBPα mutants had more modest anti-proliferative effects. Both mutants were less effective than wild-type C/EBPα in suppressing endogenous E2F-dependent transactivation and bound less E2F-2 and/or E2F-3 proteins in anti-C/EBPα immunoprecipitates. Together, these findings suggest that mutation of S21 more than its phosphorylation inhibits the anti-proliferative effects of C/EBPα due to reduced interaction with or impaired regulation of the activity of E2F proteins. By contrast, phosphorylation of serine 21 appears to have a modest role in modulating the differentiation-inducing effects of C/EBPα in K562 cells.

In vitro anti-myeloma activity of TRAIL-expressing adipose-derived mesenchymal stem cells.

Recently, genetically modified mesenchymal stem cells (MSCs) have been exploited to deliver anti-cancer bio-drugs directly within the tumour mass. Here, we explored whether adipose-derived MSCs (AD-MSCs), engineered to express the pro-apoptotic ligand TRAIL (also known as TNFSF10), kill multiple myeloma (MM) cells and migrate towards MM cells in vitro. Different MM cell lines were assessed for their sensitivity to recombinant human (rh) TRAIL alone and in combination with the proteasome inhibitor bortezomib, which was shown to enhance the effect of rhTRAIL. TRAIL(+) -AD-MSCs were co-cultured with bortezomib-pretreated MM cells and their killing activity was evaluated in presence or absence of caspase inhibition. AD-MSC migration towards media conditioned by both myeloma cells and myeloma bone fragments was also investigated. Despite moderate MM cell sensitivity to rhTRAIL, TRAIL(+) -AD-MSCs in combination with bortezomib significantly induced myeloma cell death. This effect was associated with caspase-8 activation and abrogated by capsase inhibition. On the other hand, co-culture experiments were performed to evaluate whether unmodified AD-MSCs affect myeloma cell growth in vitro. AD-MSCs appeared ineffective on myeloma cell growth and showed migratory capacity towards MM cells in vitro. These data emphasize the anti-myeloma activity of TRAIL-engineered AD-MSCs and provide support for a future model of a cell-based approach against MM.

Combination of low doses of enzastaurin and lenalidomide has synergistic activity in B-non-Hodgkin lymphoma cell lines.

Less toxic and more active treatments are needed for indolent lymphomas as there is no curative treatment, and patients eventually die due to complications related to their disease. The purpose of the present study was to assess the antitumour activity of the combination of low doses of Enzastaurin and Lenalidomide (Revlimid) on B-lymphoma cell lines. The combination of Enzastaurin and Lenalidomide, at doses as low as 1 µM, showed strong synergism against indolent lymphomas by reducing cell growth, producing an increase in G0-G1 phase followed by significant decrease in S phase, increasing apoptosis, and inhibiting PI3K/AKT, PKC and MAPK/ERK pathways. These preclinical findings, together with promising results obtained with Lenalidomide for the treatment of non-Hodgkin lymphoma, suggest that further evaluation of the combination of Enzastaurin and Lenalidomide for the treatment of indolent lymphomas is warranted. These compounds, with a favourable toxicity profile, are not classic chemotherapeutic agents, causing severe side effects, and could be considered an example of a new innovative attempt of an anti-cancer 'soft treatment'.

Cancer Stem Cells and Cell Cycle Genes as Independent Predictors of Relapse in Non-small Cell Lung Cancer: Secondary Analysis of a Prospective Study.

PURPOSE: Cancer stem cells (CSCs) are described as resistant to chemotherapy and radiotherapy. It has been shown that CSCs influence disease-free survival in patients undergoing surgery for lung cancer (NCT04634630). We recently described an overexpression of CSCs recurrence-related genes (RG) in lung cancer. This study aims to investigate CSC frequency and RG expression as predictors of disease-free survival in lung cancer. EXPERIMENTAL DESIGN: This secondary analysis of a prospective cohort study involved 22 surgical tumor specimens from 22 patients harboring early (I-II) and locally advanced (IIIA) stages ACL and SCCL. Cell population frequency analysis of ALDHhigh (CSCs) and ALDHlow (cancer cells) was performed on each tumor specimen. In addition, RG expression was assessed for 31 target genes separately in ALDHhigh and ALDHlow populations. CSCs frequency and RG expression were assessed as predictors of disease-free survival by Cox analysis. RESULTS: CSCs frequency and RG expression were independent predictors of disease-free survival. CSC frequency was not related to disease-free survival in early-stage patients (HR = 0.84, 95%CI = 0.53-1.33, P = .454), whereas it was a risk factor for locally advanced-stage patients (HR = 1.22, 95%CI = 1.09-1.35, P = .000). RG expression-if measured in CSCs-was related to a higher risk of recurrence (HR = 1.19, 95%CI = 1.03-1.39, P = .021). The effect of RG expression measured in cancer cells on disease-free survival was lower and was not statistically significant (HR = 1.12, 95%CI = 0.94-1.33, P = .196). CONCLUSIONS: CSCs frequency and RG expression are independent predictors of relapse in lung cancer. Considering these results, CSCs and RG may be considered for both target therapy and prognosis.

The Influence of Cancer Stem Cells on the Risk of Relapse in Adenocarcinoma and Squamous Cell Carcinoma of the Lung: A Prospective Cohort Study.

PURPOSE: Lung cancer relapse may be associated with the presence of a small population of cancer stem cells (CSCs) with unlimited proliferative potential. Our study assessed the relationship between CSCs and the relapse rate in patients harboring adenocarcinoma (ADL) and squamous cell carcinoma of the lung (SCCL). EXPERIMENTAL DESIGN: This is an observational prospective cohort study (NCT04634630) assessing the influence of CSC frequency on relapse rate after major lung resection in 35 patients harboring early (I-II) (n = 21) and locally advanced (IIIA) (n = 14) ADL and SCCL. There was a 2-year enrollment period followed by a 1-year follow-up period. Surgical tumor specimens were processed, and CSCs were quantified by cytofluorimetric analysis. RESULTS: Cancer stem cells were expressed in all patients with a median of 3.1% of the primary cell culture. Primary analysis showed no influence of CSC frequency on the risk of relapse (hazard ratio [HR] = 1.05, 95% confidence interval [CI] = 0.85-1.30). At secondary analysis, patients with locally advanced disease with higher CSC frequency had an increased risk of relapse (HR = 1.26, 95% CI = 1.14-1.39), whereas this was not observed in early-stage patients (HR = 0.90, 95% CI = 0.65-1.25). CONCLUSION: No association was found between CSC and relapse rates after major lung resection in patients harboring ACL and SCCL. However, in locally advanced-stage patients, a positive correlation was observed between CSC frequency and risk of relapse. These results indicate a need for further molecular investigations into the prognostic role of CSCs at different lung cancer stages. CLINICAL TRIAL REGISTRATION: NCT04634630.