Pancreatic ductal adenocarcinomas contain a subset of exclusively tumorigenic cancer stem cells (CSCs), which are capable of repopulating the entire heterogeneous cancer cell populations and are ...highly resistant to standard chemotherapy. Here we demonstrate that metformin selectively ablated pancreatic CSCs as evidenced by diminished expression of pluripotency-associated genes and CSC-associated surface markers. Subsequently, the ability of metformin-treated CSCs to clonally expand in vitro was irreversibly abrogated by inducing apoptosis. In contrast, non-CSCs preferentially responded by cell cycle arrest, but were not eliminated by metformin treatment. Mechanistically, metformin increased reactive oxygen species production in CSC and reduced their mitochondrial transmembrane potential. The subsequent induction of lethal energy crisis in CSCs was independent of AMPK/mTOR. Finally, in primary cancer tissue xenograft models metformin effectively reduced tumor burden and prevented disease progression; if combined with a stroma-targeting smoothened inhibitor for enhanced tissue penetration, while gemcitabine actually appeared dispensable.
Patient-derived in vivo models of human cancer have become a reality, yet their turnaround time is inadequate for clinical applications. Therefore, tailored ex vivo models that faithfully ...recapitulate in vivo tumour biology are urgently needed. These may especially benefit the management of pancreatic ductal adenocarcinoma (PDAC), where therapy failure has been ascribed to its high cancer stem cell (CSC) content and high density of stromal cells and extracellular matrix (ECM). To date, these features are only partially reproduced ex vivo using organoid and sphere cultures. We have now developed a more comprehensive and highly tuneable ex vivo model of PDAC based on the 3D co-assembly of peptide amphiphiles (PAs) with custom ECM components (PA-ECM). These cultures maintain patient-specific transcriptional profiles and exhibit CSC functionality, including strong in vivo tumourigenicity. User-defined modification of the system enables control over niche-dependent phenotypes such as epithelial-to-mesenchymal transition and matrix deposition. Indeed, proteomic analysis of these cultures reveals improved matrisome recapitulation compared to organoids. Most importantly, patient-specific in vivo drug responses are better reproduced in self-assembled cultures than in other models. These findings support the use of tuneable self-assembling platforms in cancer research and pave the way for future precision medicine approaches.
Pancreatic ductal adenocarcinoma is one of the deadliest carcinomas and is characterized by highly tumorigenic and metastatic cancer stem cells (CSC). CSCs evade available therapies, which ...preferentially target highly proliferative and more differentiated progenies, leaving behind CSCs as a putative source for disease relapse. Thus, to identify potentially more effective treatment regimens, we screened established and new compounds for their ability to eliminate CSCs in primary pancreatic cancer (stem) cells in vitro and corresponding patient-derived pancreatic cancer tissue xenografts in vivo. Intriguingly, we found that in vitro treatment with the antimalarial agent chloroquine significantly decreased CSCs, translating into diminished in vivo tumorigenicity and invasiveness in a large panel of pancreatic cancers. In vivo treatment in combination with gemcitabine was capable of more effectively eliminating established tumors and improved overall survival. The inhibitory effect of chloroquine was not related to inhibition of autophagy, but was due to inhibition of CXCL12/CXCR4 signaling, resulting in reduced phosphorylation of ERK and STAT3. Furthermore, chloroquine showed potent inhibition of hedgehog signaling by decreasing the production of Smoothened, translating into a significant reduction in sonic hedgehog-induced chemotaxis and downregulation of downstream targets in CSCs and the surrounding stroma. Our study demonstrates that via to date unreported effects, chloroquine is an effective adjuvant therapy to chemotherapy, offering more efficient tumor elimination and improved cure rates. Chloroquine should be further explored in the clinical setting as its success may help to more rapidly improve the poor prognosis of patients with pancreatic cancer.
The lysyl oxidase-like protein 2 (LOXL2) contributes to tumour progression and metastasis in different tumour entities, but its role in pancreatic ductal adenocarcinoma (PDAC) has not been evaluated ...in immunocompetent in vivo PDAC models.
Towards this end, we used PDAC patient data sets, patient-derived xenograft in vivo and in vitro models, and four conditional genetically-engineered mouse models (GEMMS) to dissect the role of LOXL2 in PDAC. For GEMM-based studies,
;
;
mice (KPC) and the
;
mice (KC) were crossed with
allele floxed mice (
) or conditional
overexpressing mice (R26
) to generate KPCL2
or KCL2
and KPCL2
or KCL2
mice, which were used to study overall survival; tumour incidence, burden and differentiation; metastases; epithelial to mesenchymal transition (EMT); stemness and extracellular collagen matrix (ECM) organisation.
Using these PDAC mouse models, we show that while
ablation had little effect on primary tumour development and growth, its loss significantly decreased metastasis and increased overall survival. We attribute this effect to non-cell autonomous factors, primarily ECM remodelling.
overexpression, on the other hand, promoted primary and metastatic tumour growth and decreased overall survival, which could be linked to increased EMT and stemness. We also identified tumour-associated macrophage-secreted oncostatin M (OSM) as an inducer of LOXL2 expression, and show that targeting macrophages in vivo affects
and
expression and collagen fibre alignment.
Taken together, our findings establish novel pathophysiological roles and functions for LOXL2 in PDAC, which could be potentially exploited to treat metastatic disease.
: The 5-year survival rate for pancreatic ductal adenocarcinoma (PDAC) has remained at <5% for decades because no effective therapies have been identified. Integrin αvβ6 is overexpressed in most PDAC ...and represents a promising therapeutic target. Thus, we attempted to develop an αvβ6-specific peptide-drug conjugate (PDC) for therapy of PDAC.
: We conjugated the DNA-binding pyrrolobenzodiazepine (PBD)-based payload SG3249 (tesirine) to an αvβ6-specific 20mer peptide from the VP1 coat protein of foot-and-mouth-disease virus (FMDV) (forming conjugate SG3299) or to a non-targeting peptide (forming conjugate SG3511). PDCs were tested for specificity and toxicity on αvβ6-negative versus-positive PDAC cells, patient-derived cell lines from tumor xenografts, and on two different
models of PDAC. Immunohistochemical analyses were performed to establish therapeutic mechanism.
: The αvβ6-targeted PDC SG3299 was significantly more toxic (up to 78-fold) for αvβ6-expressing versus αvβ6-negative PDAC cell lines
, and achieved significantly higher toxicity at equal dose than the non-targeted PDC SG3511 (up to 15-fold better). Moreover, SG3299 eliminated established (100mm
) Capan-1 PDAC human xenografts, extending the lifespan of mice significantly (P=0.005). Immunohistochemistry revealed SG3299 induced DNA damage and apoptosis (increased γH2AX and cleaved caspase 3, respectively) associated with significant reductions in proliferation (Ki67), β6 expression and PDAC tumour growth.
: The FMDV-peptide drug conjugate SG3299 showed αvβ6-selectivity
and
and can specifically eliminate αvβ6-positive cancers, providing a promising new molecular- specific therapy for pancreatic cancer.
The stromal microenvironment controls response to injury and inflammation, and is also an important determinant of cancer cell behavior. However, our understanding of its modulation by miRNA (miR) ...and their respective targets is still sparse. Here, we identified the miR-25-93-106b cluster and two new target genes as critical drivers for metastasis and immune evasion of cancer cells. Using miR-25-93-106b knockout mice or antagomiRs, we demonstrated regulation of the production of the chemoattractant CXCL12 controlling bone marrow metastasis. Moreover, we identified the immune checkpoint PD-L1 (CD274) as a novel miR-93/106b target playing a central role in diminishing tumor immunity. Eventually, upregulation of miR-93 and miR-106b via miR-mimics or treatment with an epigenetic reader domain (BET) inhibitor resulted in diminished expression of CXCL12 and PD-L1. These data suggest a potential new therapeutic rationale for use of BET inhibitors for dual targeting of cancers with strong immunosuppressive and metastatic phenotypes.
Pancreatic ductal adenocarcinomas contain a subset of exclusively tumorigenic cancer stem cells (CSCs), which are capable of repopulating the entire heterogeneous cancer cell populations and are ...highly resistant to standard chemotherapy. Here we demonstrate that metformin selectively ablated pancreatic CSCs as evidenced by diminished expression of pluripotency-associated genes and CSC-associated surface markers. Subsequently, the ability of metformin-treated CSCs to clonally expand in vitro was irreversibly abrogated by inducing apoptosis. In contrast, non-CSCs preferentially responded by cell cycle arrest, but were not eliminated by metformin treatment. Mechanistically, metformin increased reactive oxygen species production in CSC and reduced their mitochondrial transmembrane potential. The subsequent induction of lethal energy crisis in CSCs was independent of AMPK/mTOR. Finally, in primary cancer tissue xenograft models metformin effectively reduced tumor burden and prevented disease progression; if combined with a stroma-targeting smoothened inhibitor for enhanced tissue penetration, while gemcitabine actually appeared dispensable.
The successful clinical application of nucleic acid-based therapeutic strategies has been limited by the poor delivery efficiency achieved by existing vectors. The development of alternative delivery ...systems for improved biological activity is, therefore, mandatory. Since the seminal observations two decades ago that the Tat protein, and derived peptides, can translocate across biological membranes, cell-penetrating peptides (CPPs) have been considered one of the most promising tools to improve non-invasive cellular delivery of therapeutic molecules. Despite extensive research on the use of CPPs for this purpose, the exact mechanisms underlying their cellular uptake and that of peptide conjugates remain controversial. Over the last years, our research group has been focused on the S4
-PV cell-penetrating peptide, a prototype of this class of peptides that results from the combination of 13-amino-acid cell penetrating sequence derived from the Dermaseptin S4 peptide with the SV40 large T antigen nuclear localization signal. By performing an extensive biophysical and biochemical characterization of this peptide and its analogs, we have gained important insights into the mechanisms governing the interaction of CPPs with cells and their translocation across biological membranes. More recently, we have started to explore this peptide for the intracellular delivery of nucleic acids (plasmid DNA, siRNA and oligonucleotides). In this review we discuss the current knowledge of the mechanisms responsible for the cellular uptake of cell-penetrating peptides, including the S4
-PV peptide, and the potential of peptide-based formulations to mediate nucleic acid delivery.
Chronic mitral regurgitation promotes left atrial (LA) remodeling. However, the significance of LA dysfunction in the setting of ventricular functional mitral regurgitation (FMR) has not been fully ...investigated. Our aim was to assess the prognostic impact of peak atrial longitudinal strain (PALS), a surrogate of LA function, in patients with FMR and reduced left ventricular ejection fraction (LVEF).
Patients with at least mild ventricular FMR and LVEF < 50% under optimized medical therapy who underwent transthoracic echocardiography at a single center were retrospectively identified in the laboratory database. PALS was assessed by 2D speckle tracking in the apical 4-chamber view and the study population was divided in two groups according to the best cut-off value of PALS, using receiver operating characteristics (ROC) curve analysis. The primary endpoint-point was all-cause mortality.
A total of 307 patients (median age 70 years, 77% male) were included. Median LVEF was 35% (IQR: 27 - 40%) and median effective regurgitant orifice area (EROA) was 15mm
(IQR: 9 - 22mm
). According to current European guidelines, 32 patients had severe FMR (10%). During a median follow-up of 3.5 years (IQR 1.4 - 6.6), 148 patients died. The unadjusted mortality incidence per 100 persons-years increased with progressively lower values of PALS. On multivariable analysis, PALS remained independently associated with all-cause mortality (adjusted hazard ratio 1.052 per % decrease; 95% CI: 1.010 - 1.095; P = 0.016), even after adjustment for several (n = 14) clinical and echocardiographic confounders.
PALS is independently associated with all-cause mortality in patients with reduced LVEF and ventricular FMR.
Proportionality of secondary mitral regurgitation (sMR) may be a key factor in deciding whether a patient may benefit from mitral intervention. The aim of this study was to evaluate the prognostic ...value of two different concepts of proportionality and assess their ability to improve MR stratification proposed by the American Society of Echocardiography (ASE) guidelines.
We conducted a retrospective analysis in patients with reduced left ventricular ejection fraction (LVEF) (<50%) and at least mild sMR. Proportionality status was calculated using formulas proposed by a) Grayburn et al. – disproportionate sMR defined as EROALVEDV >0.14; b) Lopes et al. – disproportionate sMR whenever measured EROA>theoretical EROA (determined as 50%×LVEF×LVEDVMitralVTI). Primary endpoint was all-cause mortality.
A total of 572 patients (69±12 years; 76% male) were included. Mean LVEF was 33±9%, with a median left ventricular end-diastolic volume of 174 mL 136;220 and a median effective regurgitant orifice area of 14 mm2 8;22. During mean follow-up of 4.1±2.7 years, there were 254 deaths. There was considerable disagreement (p<0.001) between both formulas: of 96 patients with disproportionate sMR according to Lopes’ criteria, 46 (48%) were considered proportionate according to Grayburn's; and of 62 patients with disproportionate sMR according to Grayburn's, 12 (19%) were considered proportionate according to Lopes’ formula.
In multivariate analysis, only Lopes’ definition of disproportionate sMR maintained independent prognostic value (hazard ratio 1.5; 95% confidence interval 1.07–2.1, p=0.018) and improved the risk stratification of ASE sMR classification.
Of the two formulas available to define disproportionate sMR, Lopes’ model emerged as the only one with independent prognostic value while improving the risk stratification proposed by the ASE guidelines.
A proporcionalidade da regurgitação mitral secundária (sMR) pode ser um fator chave na decisão de que doentes podem beneficiar de intervenção mitral. O objetivo deste estudo foi de avaliar o valor prognóstico de dois modelos de proporcionalidade e aferir a sua capacidade para melhorar a estratificação da regurgitação mitral proposta pelas guidelines da ASE.
Realizamos um estudo retrospetivo com doentes com LVEF reduzida (<50%) e pelo menos sMR ligeira. O status de proporcionalidade foi calculado usando as fórmulas propostas por: a) Grayburn et al. – sMR desproporcional definida por EROALVEDV >0.14; b) Lopes et al. – sMR desproporcional quando o EROA medido>EROA teórico (determinado por 50%×LVEF×LVEDVMitralVTI). O endpoint primário foi mortalidade por qualquer causa.
Foram incluídos 572 pacientes (69±12 anos; 76% sexo masculino). LVEF média foi de 33±9%, com um LVEDV mediano de 174 mL 136;220 e um EROA mediano de 14 mm2 8;22. Após um follow-up médio de 4,1±2,7 anos, ocorreram 254 mortes. Verificou-se marcada discordância (p<0,001) entre ambas as fórmulas: de entre 96 doentes com sMR desproporcional pelo modelo de Lopes, 46 (48%) foram considerados proporcionais pela fórmula de Grayburn; de entre os 62 doentes com sMR desproporcional pelo modelo de Grayburn, 12 (19%) foram considerados proporcionais pelo modelo de Lopes.
Em análise multivariável, apenas a definição de desproporcionalidade descrita por Lopes manteve valor prognóstico independente (HR 1,5; 95%CI 1,07–2,1, p=0,018) e melhorou a estratificação de risco pela classificação da sMR da ASE.
De entre as duas fórmulas disponíveis para definição sMR desproporcional, apenas o modelo de Lopes demonstrou valor prognóstico independente e melhorou a estratificação de risco proposta pelas guidelines da ASE.
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