Leila Akkari

Glioblastomas are aggressive primary brain tumors with an inherent resistance to T cell-centric immunotherapy due to their low mutational burden and immunosuppressive tumor microenvironment. Here we report that fractionated radiotherapy of preclinical glioblastoma models induce a tenfold increase in T cell content. Orthogonally, spatial imaging mass cytometry shows T cell enrichment in human recurrent tumors compared with matched primary glioblastoma. In glioblastoma-bearing mice, α-PD-1 treatment applied at the peak of T cell infiltration post-radiotherapy results in a modest survival benefit compared with concurrent α-PD-1 administration. Following α-PD-1 therapy, CD103+ regulatory T cells (Tregs) with upregulated lipid metabolism accumulate in the tumor microenvironment, and restrain immune checkpoint blockade response by repressing CD8+ T cell activation. Treg targeting elicits tertiary lymphoid structure formation, enhances CD4+ and CD8+ T cell frequency and function and unleas...

Purpose of Review Glioblastoma is the commonest primary brain cancer in adults whose outcomes are amongst the worst of any cancer. The current treatment pathway comprises surgery and postoperative chemoradiotherapy though unresectable diffusely infiltrative tumour cells remain untreated for several weeks post-diagnosis. Intratumoural heterogeneity combined with increased hypoxia in the postoperative tumour microenvironment potentially decreases the efficacy of adjuvant interventions and fails to prevent early postoperative regrowth, called rapid early progression (REP). In this review, we discuss the clinical implications and biological foundations of post-surgery REP. Subsequently, clinical interventions potentially targeting this phenomenon are reviewed systematically. Recent Findings Early interventions include early systemic chemotherapy, neoadjuvant immunotherapy, local therapies delivered during surgery (including Gliadel wafers, nanoparticles and stem cell therapy) and severa...

Quantitative differences in signal transduction are to date an understudied feature of tumour heterogeneity. The MAPK Erk pathway, which is activated in a large proportion of human tumours, is a prototypic example of distinct cell fates being driven by signal intensity. We have used primary hepatocyte precursors transformed with different dosages of an oncogenic form of Ras to model subclonal variations in MAPK signalling. Orthotopic allografts of Ras-transformed cells in immunocompromised mice gave rise to fast-growing aggressive tumours, both at the primary location and in the peritoneal cavity. Fluorescent labelling of cells expressing different oncogene levels, and consequently varying levels of MAPK Erk activation, highlighted the selection processes operating at the two sites of tumour growth. Indeed, significantly higher Ras expression was observed in primary as compared to metastatic tumours, despite the evolutionary trade-off of increased apoptotic death in the liver that c...

We thank Alisi and colleagues for their comments [1] on our recent report characterizing the effects of the hepatitis C virus (HCV) proteins on metabolic liver zonation and steatosis [2]. In a transgenic mouse model with hepato-specific expression of the full complement of HCV proteins we found that fatty acid synthase, a major lipogenic enzyme, was redistributed to the midzone of the lobule, coinciding with zonated accumulation of lipids. Based on these and additional results, we hypothesized that low levels of viral proteins are sufficient to drive striking alterations of hepatic metabolic zonation. However, not all genes that display zonated expression in the liver were altered in the animal model studied. Notably, we found no change in the pattern of expression of E-cadherin or arginase 1. In contrast to the results reported by Alisi et al. we did not study the global level of expression of the latter. Alisi et al. compared arginase 1 expression in liver tissues from children with no hepatic pathologies or suffering from non-alcoholic fatty liver disease (NAFLD) or hepatitis C [1]. The quantification of the immunhistochemical staining showed identical mean intensity of arginase 1 in healthy livers and in HCVpositive patients without steatosis. This is consistent with our results showing no difference of arginase 1 expression in hepatocytes from control and HCV-transgenic mice [2]. In our work, appearance of steatosis did not alter arginase distribution. In contrast, Alisi et al. observed increased arginase 1 expression in HCV patients with steatosis and in NAFLD. Their results highlight the link between lipid accumulation and arginase expression, as well as the effect of HCV infection on arginase 1 accumulation. Noteworthy, Alisi et al. give no information on the HCV genotype, while our study focused solely on genotype 1. It would be of interest to determine whether the reported observations are genotype specific or more generalizable. As Alisi and colleagues correctly pointed out, the role of arginase 1 in steatosis is not well understood and possibly underestimated. Interestingly, its increased expression has been described in HCV infection and it was suggested that it might participate in promoting cell growth and proliferation [3]. Indeed, arginase 1 converts arginine to ornithine, which is further metabolized to polyamines that promote cellular proliferation. Thus, elevated arginase 1 expression may be hepatoprotective during chronic infection by promoting survival and proliferation of HCV-infected hepatocytes. In consequence, the inflammatory immune response that contributes to liver damage would be inefficient in eliminating virus-infected cells. Elevated arginase 1 expression is associated with many tumor types [4] and therapeutic strategies aiming at inhibiting this metabolic pathway are being tested in the clinic. Interestingly, in HCC, the predominant peri-tumoral distribution of arginase 1 expression is in agreement with proteomic analyses of samples from HCV infected HCC and non-tumoral liver [3,5]. Recent evidence indicates that interactions between tumor cells and the host microenvironment have a major role in driving cancer progression and metastasis [6,7]. The abundance of macrophages, pivotal members of tumor stroma, strongly correlates with poor prognosis in different types of solid tumors, including HCC [8]. It has been proposed that tumor-associated macrophages (M2-polarized) play an important role in generating overall immunosuppressive milieu within the tumor microenvironment that suppresses anti-tumor immunity and promotes tumor progression [6]. Interestingly, the differential metabolism of L-arginine provides a means of distinguishing the two macrophage activation states. M1, or classically activated macrophages, upregulate iNOS to catabolize L-arginine to nitric oxide and citrulline, while M2, or alternatively activated macrophages, induce arginase 1, the enzyme upstream of polyamines production, and thus increase collagen synthesis and cellular proliferation [9]. In HCC, high numbers of peri-tumoral M2-polarized TAMs are associated with poor patient prognosis, and while no analyses of arginase 1 expression have yet been conducted in this context, it is likely that they express high levels of this enzyme along with the other M2 genes described. In this context, TAM-derived arginase 1 might promote tumorigenesis in a non-cell autonomous manner. Furthermore, several TAM-derived factors, such as

Hepatocellular carcinoma (HCC), a malignancy that arises in the context of a damaged liver, is the second leading cause of cancer-related mortality worldwide, in part due to limited effective treatment and the rapid development of resistance. While many studies have focused on the cellular changes that drive resistance to therapy, little attention has been given to the role that liver tumor microenvironment plays in this process. Liver fibrosis, extracellular matrix accumulation, angiogenesis, hypoxia, and inflammation are factors that directly contribute to liver tumorigenesis and can also promote resistance. Understanding the interactions of the components of the liver tumor microenvironment with cancer cells will be critical to design strategies to overcome resistance and improve the therapeutic efficacy of current and future therapies.

Leila Akkari began her independent career in 2017 as an Assistant Professor at the Netherlands Cancer Institute in Amsterdam after working at Memorial Sloan Kettering Cancer Center in NYC. Two years ago, she was selected as one of the junior members of the Oncode Institute, a virtual group of cancer research labs based on the Netherlands. In this short Q&A, she tells us about her research and how her diverse background has helped her as a scientist. Dr. Akkari also shares some great pointers on the biggest hurdles women in STEM face and tips to overcome them.

Tumor-associated macrophages (TAMs) and microglia (MG) are potent regulators of glioma development and progression. However, the dynamic alterations of distinct TAM populations during the course of therapeutic intervention, response, and recurrence have not yet been fully explored. Here, we investigated how radiotherapy changes the relative abundance and phenotypes of brain-resident MG and peripherally recruited monocyte-derived macrophages (MDMs) in glioblastoma. We identified radiation-specific, stage-dependent MG and MDM gene expression signatures in murine gliomas and confirmed altered expression of several genes and proteins in recurrent human glioblastoma. We found that targeting these TAM populations using a colony-stimulating factor–1 receptor (CSF-1R) inhibitor combined with radiotherapy substantially enhanced survival in preclinical models. Our findings reveal the dynamics and plasticity of distinct macrophage populations in the irradiated tumor microenvironment, which has...

Lipid accumulation within the lumen of endolysosomal vesicles is observed in various pathologies including atherosclerosis, liver disease, neurological disorders, lysosomal storage disorders, and cancer. Current methods cannot measure lipid flux specifically within the lysosomal lumen of live cells. We developed an optical reporter, composed of a photoluminescent carbon nanotube of a single chirality, which responds to lipid accumulation via modulation of the nanotube's optical bandgap. The reporter localizes exclusively to the lysosomal lumen where the emission wavelength can be spatially resolved to generate quantitative maps of lipid content. Measurements of patient-derived Niemann-Pick type C fibroblasts identified lipid accumulation and phenotypic reversal of this lysosomal storage disease. The reporter discerned sub-cellular differences in lipid content, illuminating significant intracellular heterogeneity among lysosomes of differentiating bone marrow-derived monocytes. S...

Obesity is associated with chronic, low-grade inflammation, which can disrupt homeostasis within tissue microenvironments. Given the correlation between obesity and relative risk of death from cancer, we investigated whether obesity-associated inflammation promotes metastatic progression. We demonstrate that obesity causes lung neutrophilia in otherwise normal mice, which is further exacerbated by the presence of a primary tumour. The increase in lung neutrophils translates to increased breast cancer metastasis to this site, in a GM-CSF- and IL5-dependent manner. Importantly, weight loss is sufficient to reverse this effect, and reduce serum levels of GM-CSF and IL5 in both mouse models and humans. Our data indicate that special consideration of the obese patient population is critical for effective management of cancer progression.

The genetic and phenotypic diversity of cells within tumors is a major obstacle for cancer treatment. Because of the stochastic nature of genetic alterations, this intratumoral heterogeneity is often viewed as chaotic. Here we show that the altered metabolism of cancer cells creates predictable gradients of extracellular metabolites that orchestrate the phenotypic diversity of cells in the tumor microenvironment. Combining experiments and mathematical modeling, we show that metabolites consumed and secreted within the tumor microenvironment induce tumor-associated macrophages (TAMs) to differentiate into distinct subpopulations according to local levels of ischemia and their position relative to the vasculature. TAMs integrate levels of hypoxia and lactate into progressive activation of MAPK signaling that induce predictable spatial patterns of gene expression, such as stripes of macrophages expressing arginase 1 (ARG1) and mannose receptor, C type 1 (MRC1). These phenotypic changes...

Deregulated cathepsin proteolysis occurs across numerous cancers, but in vivo substrates mediating tumorigenesis remain ill-defined. Applying 8-plex iTRAQ terminal amine isotopic labeling of substrates (TAILS), a systems-level N-terminome degradomics approach, we identified cathepsin B, H, L, S, and Z in vivo substrates and cleavage sites with the use of six different cathepsin knockout genotypes in the Rip1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis. Among 1,935 proteins and 1,114 N termini identified by TAILS, stable proteolytic products were identified in wild-type tumors compared with one or more different cathepsin knockouts (17%-44% of 139 cleavages). This suggests a lack of compensation at the substrate level by other cathepsins. The majority of neo-N termini (56%-83%) for all cathepsins was consistent with protein degradation. We validated substrates, including the glycolytic enzyme pyruvate kinase M2 associated with the Warburg effect, the ER chaperone GRP7...

Pleuropulmonary synovial sarcoma (PPSS) is an extremely rare malignant tumor, which is increasingly recognized as a subtype of sarcoma with a distinctive chromosomal translocation specific to synovial sarcoma. It is often presents like any thoracic tumor with symptoms such as chest pain or cough. Here we report a case of PPSS in a 49-year-old woman presenting with cough, shortness of breath and chest pain. And who were found upon histologic examination of the resection specimen to have cystic primary pleuropulmonary synovial sarcoma.

Macrophages accumulate with glioblastoma multiforme (GBM) progression and can be targeted via inhibition of colony-stimulating factor-1 receptor (CSF-1R) to regress high-grade tumors in animal models of this cancer. However, whether and how resistance emerges in response to sustained CSF-1R blockade is unknown. We show that although overall survival is significantly prolonged, tumors recur in >50% of mice. Gliomas reestablish sensitivity to CSF-1R inhibition upon transplantation, indicating that resistance is tumor microenvironment-driven. Phosphatidylinositol 3-kinase (PI3K) pathway activity was elevated in recurrent GBM, driven by macrophage-derived insulin-like growth factor-1 (IGF-1) and tumor cell IGF-1 receptor (IGF-1R). Combining IGF-1R or PI3K blockade with CSF-1R inhibition in recurrent tumors significantly prolonged overall survival. Our findings thus reveal a potential therapeutic approach for treating resistance to CSF-1R inhibitors.

Proteases are important for regulating multiple tumorigenic processes, including angiogenesis, tumor growth, and invasion. Elevated protease expression is associated with poor patient prognosis across numerous tumor types. Several multigene protease families have been implicated in cancer, including cysteine cathepsins. However, whether individual family members have unique roles or are functionally redundant remains poorly understood. Here we demonstrate stage-dependent effects of simultaneously deleting cathepsin B (CtsB) and CtsS in a murine pancreatic neuroendocrine tumor model. Early in tumorigenesis, the double knockout results in an additive reduction in angiogenic switching, whereas at late stages, several tumorigenic phenotypes are unexpectedly restored to wild-type levels. We identified CtsZ, which is predominantly supplied by tumor-associated macrophages, as the compensatory protease that regulates the acquired tumor-promoting functions of lesions deficient in both CtsB and CtsS. Thus, deletion of multiple cathepsins can lead to stage-dependent, compensatory mechanisms in the tumor microenvironment, which has potential implications for the clinical consideration of selective versus pan-family cathepsin inhibitors in cancer.

Drastic metabolic alterations, such as the Warburg effect, are found in most if not all types of malignant tumors. Emerging evidence shows that cancer cells benefit from these alterations, but little is known about how they affect noncancerous stromal cells within the tumor microenvironment. Here we show that cancer cells are better adapted to metabolic changes in the microenvironment, leading to the emergence of spatial structure. A clear example of tumor spatial structure is the localization of tumor-associated macrophages (TAMs), one of the most common stromal cell types found in tumors. TAMs are enriched in well-perfused areas, such as perivascular and cortical regions, where they are known to potentiate tumor growth and invasion. However, the mechanisms of TAM localization are not completely understood. Computational modeling predicts that gradients--of nutrients, gases, and metabolic by-products such as lactate--emerge due to altered cell metabolism within poorly perfused tumors, creating ischemic regions of the tumor microenvironment where TAMs struggle to survive. We tested our modeling prediction in a coculture system that mimics the tumor microenvironment. Using this experimental approach, we showed that a combination of metabolite gradients and differential sensitivity to lactic acid is sufficient for the emergence of macrophage localization patterns in vitro. This suggests that cancer metabolic changes create a microenvironment where tumor cells thrive over other cells. Understanding differences in tumor-stroma sensitivity to these alterations may open therapeutic avenues against cancer.

Expression of developmental genes Twist1 and Twist2 is reactivated in many human tumors. Among their oncogenic activities, induction of epithelial to mesenchymal transition is believed to increase cell motility and invasiveness and may be related to acquisition of cancer stem cell phenotype. In addition, Twist proteins promote malignant conversion by overriding two oncogene-induced failsafe programs: senescence and apoptosis. Reactive oxygen species (ROS) are also important mediators of apoptosis, senescence and motility and are tightly linked to disease, notably to cancer. We report here that Twist factors and ROS are functionally linked. In wild type cells both Twist1 and Twist2 exhibit antioxidant properties. We show that Twist-driven modulation of oncogene-induced apoptosis is linked to its effects on oxidative stress. Finally, we identify several targets that mediate Twist antioxidant activity. These findings unveil a new function of Twist factors that could be important in explaining their pleiotropic role during carcinogenesis.

Apicobasal polarity, which is essential for epithelial structure and function, is targeted by several tumour-related pathogens and is generally perturbed in the course of carcinogenesis. Hepatitis C virus (HCV) infection is associated with a strong risk of hepatocellular carcinoma, typically preceded by dysplastic alterations of cell morphology. We investigated the molecular mechanisms and the functional consequences of HCV-driven perturbations of epithelial polarity. We used biochemical, genetic, and cell biology approaches to assess the impact of hepatitis C viral protein NS5A on the polarity and function of hepatocytes and hepatic progenitors. Transgenic animals and xenograft models served for in vivo validation of the results obtained in cell culture. We found that expression of HCV-NS5A in primary hepatic precursors and in immortalized hepatocyte cell lines gave rise to profound modifications of cell polarity, leading to epithelial to mesenchymal transition (EMT). NS5A, either alone or in the context of the full complement of viral proteins in the course of infection, acted through activating Twist2, a transcriptional regulator of EMT. The effects of NS5A were additive to those of TGF-β, a cytokine abundant in diseased liver and highly relevant to HCV-related pathology. Moreover, NS5A cooperates with oncogenic Ras, giving rise to transformed, invasive cells that are highly tumorigenic in vivo. Our data suggest that in the context of HCV infection, NS5A favors formation of preneoplastic lesions by disrupting cell polarity and additional oncogenic events cooperate with the viral protein to give rise to motile and invasive tumour cells.

Cellular differentiation relies on both physical and chemical environmental cues. The bipotential mouse embryonic liver (BMEL) cells are early progenitors of liver epithelial cells with an apparently infinite proliferative potential. These cells, which remain undifferentiated in a monolayer culture, differentiate upon release from geometrical constraints imposed by growth on a stiff plastic plate. In a complex three dimensional environment of a Matrigel extracellular matrix, BMEL cells form two types of polarized organoids of distinct morphologies: cyst-like structures suggesting cholangiocyte-type organization or complex organoids, reminiscent of liver parenchyma and associated with acquisition of hepatocyte-specific phenotypic markers. The choice of the in vitro differentiation lineage is governed by Transforming Growth Factor-beta (TGF-beta) signaling. Our results suggest that morphological cues initiate the differentiation of early hepatic precursors and confirm the inhibitory role of TGF-beta on hepatocytic lineage differentiation.