Targeting Cancer Stem Cells to Overcome Chemoresistance.

Cancers are heterogeneous at the cell level, and the mechanisms leading to cancer heterogeneity could be clonal evolution or cancer stem cells. Cancer stem cells are resistant to most anti-cancer treatments and could be preferential targets to reverse this resistance, either targeting stemness pathways or cancer stem cell surface markers. Gold nanoparticles have emerged as innovative tools, particularly for photo-thermal therapy since they can be excited by laser to induce hyperthermia. Gold nanoparticles can be functionalized with antibodies to specifically target cancer stem cells. Preclinical studies using photo-thermal therapy have demonstrated the feasibility of targeting chemo-resistant cancer cells to reverse clinical chemoresistance. Here, we review the data linking cancer stem cells and chemoresistance and discuss the way to target them to reverse resistance. We particularly focus on the use of functionalized gold nanoparticles in the treatment of chemo-resistant metastatic cancers.

Differential MicroRNA Expression Levels in Cutaneous Acute Graft-Versus-Host Disease.

Allogeneic hematopoietic stem cell transplantation is a curative treatment for numerous hematological malignancies. However, acute graft-versus-host disease (aGvHD) is a major complication affecting 40-70% of all transplant patients, whereby the earliest and most frequent presentation is in the skin. MicroRNAs play a role in varied biological process and have been reported as potential biomarkers for aGvHD. More recently, microRNAs have received added attention as circulatory biomarkers that can be detected in biofluids. In this study, we performed global microRNA expression profiling using a discovery cohort of diagnostic cutaneous aGvHD biopsies (n = 5, stages 1-3) and healthy volunteers (n = 4), in order to identify a signature list of microRNAs that could be used as diagnostic biomarkers for cutaneous aGvHD. Candidate microRNAs (n = 8) were then further investigated in a validation cohort of post-HSCT skin biopsies (n = 17), pre-HSCT skin biopsies (n = 6) and normal controls (n = 6) for their association with aGvHD. Expression of let-7c (p = 0.014), miR-503-5p (p = 0.003), miR-365a-3p (p = 0.02), miR-34a-5p (p < 0.001) and miR-34a-3p (p = 0.006) were significantly differentially expressed between groups and significantly associated with survival outcome in post-HSCT patients (miR-503-5p ROC AUC = 0.83 p = 0.021, Log Rank p = 0.003; miR-34a-3p ROC AUC = 0.93, p = 0.003, Log Rank p = 0.004). There was no association with relapse. A statistical interaction between miR-34a-3p and miR-503-5p (p = 0.016) was diagnostic for aGvHD. Expression levels of the miR-34a-5p protein target p53 were assessed in the epidermis of the skin, and an inverse correlation was identified (r2 = 0.44, p = 0.039). Expression of the validated candidate microRNAs was also assessed at day 28 post-HSCT in the sera of transplant recipients, in order to investigate their potential as circulatory microRNA biomarkers. Expression of miR-503-5p (p = 0.001), miR-34a-5p (p = 0.005), and miR-34a-3p (p = 0.004) was significantly elevated in the sera of patients who developed aGvHD versus no-aGvHD (n = 30) and miR-503-5p was associated with overall survival (OS) (ROC AUC = 0.80, p = 0.04, Log Rank p = 0.041). In conclusion, this investigation reports that microRNA expression levels in clinical skin biopsies, obtained at the time of cutaneous aGvHD onset, show potential as diagnostic biomarkers for aGvHD and as predictive biomarkers for OS. In addition, the same microRNAs can be detected in the circulation and show predictive association with post-HSCT outcomes.

Targeting autophagic cancer stem-cells to reverse chemoresistance in human triple negative breast cancer.

There is growing evidence for the role of cancer stem-cells in drug resistance, but with few in situ studies on human tumor samples to decipher the mechanisms by which they resist anticancer agents.Triple negative breast cancer (TNBC) is the most severe sub-type of breast cancer, occurring in younger women and associated with poor prognosis even when treated at a localized stage.We investigated here the relationship between complete pathological response after chemotherapy and breast cancer stem-cell characteristics in pre-treatment biopsies of 78 women with triple negative breast carcinoma (TNBC).We found that chemoresistance was associated with large numbers of breast cancer stem-cells, and that these cancer stem-cells were neither proliferative nor apoptotic, but in an autophagic state related to hypoxia. Using relevant pharmacological models of patient-derived TNBC xenografts, we further investigated the role of autophagy in chemoresistance of breast cancer stem-cells. We demonstrated that hypoxia increased drug resistance of autophagic TNBC stem-cells, and showed that molecular or chemical inhibition of autophagic pathway was able to reverse chemoresistance.Our results support breast cancer stem-cell evaluation in pre-treatment biopsies of TNBC patients, and the need for further research on autophagy inhibition to reverse resistance to chemotherapy.

Primary mediastinal large B-cell lymphoma: transcriptional regulation by miR-92a through FOXP1 targeting.

BACKGROUND: Primary mediastinal large B-cell lymphoma (PMBL) shares pathological features with diffuse large B-cell lymphoma (DLBCL), and molecular features with classical Hodgkin lymphoma (cHL). The miR-17~92 oncogenic cluster, located at chromosome 13q31, is a region that is amplified in DLBCL. METHODS: Here we compared the expression of each member of the miR-17~92 oncogenic cluster in samples from 40 PMBL patients versus 20 DLBCL and 20 cHL patients, and studied the target genes linked to deregulated miRNA in PMBL. RESULTS: We found a higher level of miR-92a in PMBL than in DLBCL, but not in cHL. A combination of in silico prediction and transcriptomic analyses enabled us to identify FOXP1 as a main miR-92a target gene in PMBL, a result so far not established. This was confirmed by 3'UTR, and RNA and protein expressions in transduced cell lines. In vivo studies using the transduced cell lines in mice enabled us to demonstrate a tumor suppressor effect of miR-92a and an oncogenic effect of FOXP1.A higher expression of miR-92a and the down-regulation of FOXP1 mRNA and protein expression were also found in human samples of PMBL, while miR-92a expression was low and FOXP1 was high in DLBCL. CONCLUSIONS: We concluded to a post-transcriptional regulation by miR-92a through FOXP1 targeting in PMBL, with a clinico-pathological relevance for better characterisation of PMBL.

Donor-derived stem-cells and epithelial mesenchymal transition in squamous cell carcinoma in transplant recipients.

BACKGROUND: Skin squamous-cell-carcinoma (SCC), is the main complication in long-term kidney-transplant recipients, and it can include donor-derived cells. Preclinical models demonstrated the involvement of epithelial mesenchymal transition (EMT) in the progression of skin SCC, and the role of Snail, an EMT transcription factor, in cancer stem-cell survival and expansion.Here, we studied stem-cells and EMT expression in SCCs and concomitant actinic keratoses (AK) in kidney-transplant recipients. METHODS: In SCC and AK in 3 female recipients of male kidney-transplants, donor-derived Y chromosome in epidermal stem cells was assessed using combined XY-FISH/CD133 immunostaining, and digital-droplet-PCR on laser-microdissected CD133 expressing epidermal cells.For EMT study, double immunostainings of CD133 with vimentin or snail and slug, electron microscopy and immunostainings of keratinocytes junctions were performed. Digital droplet PCR was used to check CDH1 (E-cadherin) expression level in laser-microdissected cells co-expressing CD133 and vimentin or snail and slug.The numbers of Y-chromosome were assessed using digital droplet PCR in laser-microdissected cells co-expressing CD133 and vimentin, or snail and slug, and in CD133 positive cells not expressing any EMT maker. RESULTS: We identified donor-derived stem-cells in basal layers and invasive areas in all skin SCCs and in concomitant AKs, but not in surrounding normal skin.The donor-derived stem-cells expressed the EMT markers, vimentin, snail and slug in SCCs but not in AKs. The expression of the EMT transcription factor, SNAI1, was higher in stem-cells when they expressed vimentin. They were located in invasive areas of SCCs. In these areas, the expressions of claudin-1 and desmoglein 1 were reduced or absent, and within the basal layer there were features of basal membrane disappearance.Donor-derived stem cells were in larger numbers in stem cells co-expressing vimentin or snail and slug than in stem cells not expressing any EMT marker. CONCLUSIONS: We identified here donor-derived stem cells within skin SCC in kidney-transplant recipients. They were located in invasive areas of SCC and had EMT characteristics.

Stem cells increase in numbers in perinecrotic areas in human renal cancer.

PURPOSE: Developing strategies to overcome resistance to sunitinib is a major challenge in human renal cell carcinoma (RCC). We hypothesized that sunitinib-induced tumor necrosis-associated hypoxia could interact with renal cancer stem cells in patients with metastatic RCC. EXPERIMENTAL DESIGN: We studied tissue samples from 7 patients with primary metastatic RCC, before and after sunitinib treatment, and from six xenograft models derived from human RCC. Two xenograft models were responders to sunitinib, the four others were nonresponders. CD133/CXCR4-coexpressing cells derived from the two responder xenograft models were used for in vitro studies. RESULTS: In the seven primary RCCs, we identified a significantly larger number of CD133/CXCR4-coexpressing cells in perinecrotic versus perivascular areas. Their numbers also significantly increased after treatment, in perinecrotic areas. We reproduced these clinical and pathologic results in all six RCC xenograft models with again a preferential perinecrotic distribution of CD133-expressing cells. Necrosis occurred at day 7 in the two responder models treated with sunitinib, whereas it occurred at day 21 in the untreated controls and in the four nonresponder models. Strikingly, when we studied the six RCC xenograft models at the time necrosis, whether spontaneous or sunitinib-induced, occurred, necrosis area correlated with stem-cell number in all 120 xenografted RCCs. When studied under experimental hypoxia, the number of CD133/CXCR4-coexpressing cells and their tumorigenic potency increased whereas their sensitivity to sunitinib decreased. CONCLUSIONS: In human RCC, sunitinib was able to generate resistance to its own therapeutic effect via induced hypoxia in perinecrotic areas where cancer stem cells were found in increased numbers.

Assessment of chimerism in epithelial cancers in transplanted patients.

Cancer is now the most severe complication in the long term in transplant recipients. As most solid-organ or hematopoietic stem-cell transplantations are allogeneic, chimerism studies can be performed on cancers occurring in recipients. We summarize here the different methods used to study chimerism in cancers developing in allogeneic-transplant recipients, analyze their respective advantages and report the main results obtained from these studies. Chimerism analyses of cancers in transplant recipients require methods suited to tissue samples. In the case of gender-mismatched transplantation, the XY chromosomes can be explored using fluorescent in situ hybridization on whole-tissue sections or Y-sequence-specific PCR after the laser microdissection of tumor cells. For cancers occurring after gender-matched transplantation, laser microdissection of tumor cells enables studies of microsatellite markers and high-resolution melting analysis of mitochondrial DNA on genes with marked polymorphism, provided these are different in the donor and the recipient. The results of different studies address the cancers that develop in both recipients and in transplants. The presence of chimeric cells in these two types of cancer implies an exchange of progenitor/stem-cells between transplant and recipient, and the plasticity of these progenitor/stem-cells contributes to epithelial cancers. The presence of chimeric cells in concomitant cancers and preneoplastic lesions implies that the oncogenesis of these cancers progresses through a multistep process.

Human skin carcinoma arising from kidney transplant-derived tumor cells.

Tumor cells with donor genotype have been identified in human skin cancer after allogeneic transplantation; however, the donor contribution to the malignant epithelium has not been established. Kidney transplant recipients have an increased risk of invasive skin squamous cell carcinoma (SCC), which is associated with accumulation of the tumor suppressor p53 and TP53 mutations. In 21 skin SCCs from kidney transplant recipients, we systematically assessed p53 expression and donor/recipient origin in laser-microdissected p53+ tumor cells. In one patient, molecular analyses demonstrated that skin tumor cells had the donor genotype and harbored a TP53 mutation in codon 175. In a kidney graft biopsy performed 7 years before the skin SCC diagnosis, we found p53+ cells in the renal tubules. We identified the same TP53 mutation in these p53+ epithelial cells from the kidney transplant. These findings provide evidence for a donor epithelial cell contribution to the malignant skin epithelium in the recipient in the setting of allogeneic kidney transplantation. This finding has theoretical implications for cancer initiation and progression and clinical implications in the context of prolonged immunosuppression and longer survival of kidney transplant patients.

BCL2 expression in CD105 positive neoangiogenic cells and tumor progression in angioimmunoblastic T-cell lymphoma.

The angiogenic microenvironment has been known to be a component of angioimmunoblastic T-cell lymphoma since its initial characterization. We have shown that angioimmunoblastic T-cell lymphoma endothelial cells produce vascular endothelial growth factor-A (VEGFA), and participate in lymphoma progression. In squamous cell carcinoma, endothelial BCL2 expression induces a crosstalk with tumor cells through VEGFA, a major mediator of tumoral angiogenesis. In the present study, we analyzed BCL2 and VEGFA in 30 angioimmunoblastic T-cell lymphomas, using triple immunofluorescence to identify protein coexpression in well-characterized lymphoma cells and microenvironment neoangiogenic endothelial cells. Using quantitative real-time PCR, we assessed mRNA expression levels in laser-microdissected endothelial and lymphoma cells. In lymphoma cells, as in endothelial cells, BCL2 and VEGFA proteins were coexpressed. BCL2 was expressed only in neoangiogenic CD34(+)CD105(+) endothelial cells. In laser-microdissected cells, mRNA studies showed a significant relationship between BCL2 and VEGFA levels in CD34(+) endothelial cells, but not in CD3(+)CD10(+)lymphoma cells, or in CD34(+) endothelial cells from lymph node hyperplasia. Further study showed that, in AITL, BCL2 mRNA levels in CD34(+)CD105(+) neoangiogenic endothelial cells also correlated with microvessel density, International Prognostic Index, Ann Arbor stage, bone marrow involvement and elevated LDH. BCL2 expression by CD105(+) neoangiogenic endothelial cells is related to tumor progression in angioimmunoblastic T-cell lymphoma.

Protection against myocardial infarction and no-reflow through preservation of vascular integrity by angiopoietin-like 4.

BACKGROUND: Increased permeability, predominantly controlled by endothelial junction stability, is an early event in the deterioration of vascular integrity in ischemic disorders. Hemorrhage, edema, and inflammation are the main features of reperfusion injuries, as observed in acute myocardial infarction (AMI). Thus, preservation of vascular integrity is fundamental in ischemic heart disease. Angiopoietins are pivotal modulators of cell-cell junctions and vascular integrity. We hypothesized that hypoxic induction of angiopoietin-like protein 4 (ANGPTL4) might modulate vascular damage, infarct size, and no-reflow during AMI. METHODS AND RESULTS: We showed that vascular permeability, hemorrhage, edema, inflammation, and infarct severity were increased in angptl4-deficient mice. We determined that decrease in vascular endothelial growth factor receptor 2 (VEGFR2) and VE-cadherin expression and increase in Src kinase phosphorylation downstream of VEGFR2 were accentuated after ischemia-reperfusion in the coronary microcirculation of angptl4-deficient mice. Both events led to altered VEGFR2/VE-cadherin complexes and to disrupted adherens junctions in the endothelial cells of angptl4-deficient mice that correlated with increased no-reflow. In vivo injection of recombinant human ANGPTL4 protected VEGF-driven dissociation of the VEGFR2/VE-cadherin complex, reduced myocardial infarct size, and the extent of no-reflow in mice and rabbits. CONCLUSIONS: These data showed that ANGPTL4 might constitute a relevant target for therapeutic vasculoprotection aimed at counteracting the effects of VEGF, thus being crucial for preventing no-reflow and conferring secondary cardioprotection during AMI.

IDO in human gut graft-versus-host disease.

Although rodent graft-versus-host disease (GVHD) models have suggested that indoleamine 2,3-dioxygenase (IDO) is a critical regulator of gastrointestinal GVHD, parallel human studies on IDO expression have not been reported. IDO expression was assessed in 20 patients who underwent duodenal biopsy. IDO was upregulated in epithelial cells. In situ analyses reveal that macrophages and dendritic cells stain positive for IDO, but that most of the IDO(+) cells were a novel population of CD3(+)CD4(+)IDO(+) cells. The proportion of CD4(+)IDO(+) T cells was significantly higher in patients with moderate GVHD. In situ regulatory T cell and Th17 numbers correlated with overall severity. Although needing confirmatory results from larger sample sets, these data are consistent with the hypothesis that IDO is involved in regulating gastrointestinal GVHD.

Endothelial damage in all types of T-lymphocyte-mediated drug-induced eruptions.

BACKGROUND: In severe drug-induced eruptions, bullous lesions can be associated with immune complex-mediated vasculitis and/or with T-lymphocyte-mediated keratinocyte apoptosis. We have recently identified endothelial cell apoptosis in severe bullous T-lymphocyte-mediated drug-induced eruptions. We assessed microvessel involvement in the whole spectrum of T-lymphocyte-mediated drug-induced eruptions. Thirty-two patients with T-lymphocyte-mediated drug-induced eruptions in 4 groups (8 cases of toxic epidermal necrolysis/Stevens-Johnson syndrome, 8 cases of drug rash with eosinophilia and systemic symptoms, 8 cases of acute generalized exanthematous pustulosis, 8 cases of drug maculopapular exanthema) and 8 healthy controls were included. On skin biopsy specimens, we performed a systematic ultrastructural study of endothelial cells, vascular walls, and inflammatory cells; a quantification of apoptotic cells, inflammatory infiltrate, and immune complex deposits; and we assessed granzyme-B, tumor necrosis factor, and Fas ligand expression. Correlations of apoptosis with clinical data of skin lesions and systemic involvement in liver, kidney, lung, and lymph nodes were then assessed. OBSERVATIONS: Findings from ultrastructural study showed that endothelial cell apoptosis was present in all 32 drug-induced eruptions. No leukocytoclastic vasculitis was associated. Granzyme-B and tumor necrosis factor were expressed around microvessels. In toxic epidermal necrolysis/Stevens-Johnson syndrome and drug rash with eosinophilia and systemic symptoms, the number of apoptotic endothelial cells was related to the extension of skin lesions and the presence of purpura. It was also related to liver and kidney involvement. CONCLUSIONS: Endothelial apoptosis occurs in skin microvessels of all types of T-lymphocyte-mediated drug-induced eruptions. This skin endothelial cell damage is related to the severity of skin lesions and systemic involvement.

Aflibercept-mediated early angiogenic changes in aggressive B-cell lymphoma.

PURPOSE: Diffuse large B-cell lymphoma is the most common and one of the most aggressive lymphomas in adults. The standard, R-CHOP-based treatment needs improvement. There has been recent interest in anti-angiogenic therapy, but its cellular effects in lymphoma are little known. METHODS: In five aggressive B-cell lymphoma patients, we analyzed the microvessel and lymphoma cell changes after Aflibercept, an angiogenic inhibitor. Under ultrasonography, we performed two biopsies, one before any treatment and one two hours after Aflibercept, before R-CHOP. Using ultrasonography, immuno-histochemistry, double immuno-fluorescent staining, and electron microscopy, we compared the early changes induced by Aflibercept to the early changes induced by R-CHOP in three control patients. RESULTS: We identified microvessel damage in the five patients treated with Aflibercept but not in the three patients treated with R-CHOP. Two hours after Aflibercept, microvessel damage was focal, with severely damaged microvessel sections close to normal ones in the same area; different stages of microvessel damage were concomitantly found, with an increase in relative necrosis area in three cases. There was no difference in necrosis or relative microvessel area after R-CHOP. For lymphoma cells, the two biotherapies induced similar changes, with increase in apoptosis but not in proliferation. CONCLUSION: We identified focal microvascular damage, necrosis, and apoptosis of lymphoma cells in aggressive B-cell lymphoma as soon as 2 h after Aflibercept. This suggests that there is more than one mechanism associated with the early effect of anti-angiogenic therapy in lymphoma.

Profile of macrophages in human abdominal aortic aneurysms: a transcriptomic, proteomic, and antibody protein array study.

Abdominal aortic aneurysms (AAA) are defined by an increased aortic diameter and characterized by impairment of the extracellular matrix, macrophages infiltration and decreased density of smooth muscle cells. Our aim is to identify the key molecules involved in the pathogenesis of AAAs. This study investigated transcriptomic and proteomic profiles of macrophages from AAA patients (>50 mm aortic diameter) (n = 24) and peripheral arterial occlusion (PAO) patients without AAA detected (n = 18), who both needed a surgery. An antibody protein microarray, generated by printing antibodies onto membranes against proteins selected from the transcriptomic and proteomic analysis, was performed to validate the proteins differentially expressed specifically in macrophages and plasma from the same patients. We found a restricted number of proteins differentially expressed between AAA and PAO patients: TIMP-3, ADAMTS5, and ADAMTS8 that differ significantly in plasma of AAA patients compared to PAO patients, as found in the macrophages. In contrast to plasma MMP-9, soluble glycoprotein V (sGPV) and plasmin-antiplasmin complex levels, plasma TIMP-3 levels were not correlated to AAA size but interestingly correlated to sGPV, a platelet activation marker. Combining transcriptomic and proteomic is a valid approach to identify diseases causing proteins and potential biomarkers.

Th17/Treg ratio in human graft-versus-host disease.

Th17 cells have never been explored in human graft-versus-host disease (GVHD). We studied the correlation between the presence of Th17 cells with histologic and clinical parameters. We first analyzed a cohort of 40 patients with GVHD of the gastrointestinal tract. Tumor necrosis factor (TNF), TNF receptors, and Fas expression, and apoptotic cells, CD4(+)IL-17(+) cells (Th17), and CD4(+)Foxp3(+) cells (Treg) were quantified. A Th17/Treg ratio less than 1 correlated both with the clinical diagnosis (P < .001) and more than 2 pathologic grades (P < .001). A Th17/Treg ratio less than 1 also correlated with the intensity of apoptosis of epithelial cells (P = .03), Fas expression in the cellular infiltrate (P = .003), TNF, and TNF receptor expression (P < .001). We then assessed Th17/Treg ratio in 2 other independent cohorts; a second cohort of 30 patients and confirmed that Th17/Treg ratio less than 1 correlated with the pathologic grade of GI GVHD. Finally, 15 patients with skin GVHD and 11 patients with skin rash but without pathologic GVHD were studied. Results in this third cohort of patients with skin disease confirmed those found in patients with GI GVHD. These analyses in 96 patients suggest that Th17/Treg ratio could be a sensitive and specific pathologic in situ biomarker of GVHD.