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High-grade gliomas, the most common and aggressive primary brain tumors, are characterized by a complex tumor microenvironment (TME). Among the immune cells infiltrating the glioma TME, tumor-associated microglia and macrophages (TAMs) constitute the major compartment. In patients with gliomas, increased TAM abundance is associated with more aggressive disease. Alterations in TAM phenotypes and functions have been reported in preclinical models of multiple cancers during tumor development and after therapeutic interventions, including radiotherapy and molecular targeted therapies. These findings indicate that it is crucial to evaluate TAM abundance and dynamics over time. Current techniques to quantify TAMs in patients rely mainly on histological staining of tumor biopsies. Although informative, these techniques require an invasive procedure to harvest the tissue sample and typically only result in a snapshot of a small region at a single point in time. Fluorine isotope 19 MRI ( 19 F MRI) represents a powerful means to noninvasively and longitudinally monitor myeloid cells in pathological conditions by intravenously injecting perfluorocarbon-containing nanoparticles (PFC-NP). In this study, we demonstrated the feasibility and power of 19 F MRI in preclinical models of gliomagenesis, breast-to-brain metastasis, and breast cancer and showed that the major cellular source of 19 F signal consists of TAMs. Moreover, multispectral 19 F MRI with two different PFC-NP allowed us to identify spatially and temporally distinct TAM niches in radiotherapy-recurrent murine gliomas. Together, we have imaged TAMs noninvasively and longitudinally with integrated cellular, spatial, and temporal resolution, thus revealing important biological insights into the critical functions of TAMs, including in disease recurrence.

Tumors evolve in a dynamic communication with their native tissue environment and recruited immune cells. The diverse components of the tumor microenvironment (TME) can critically regulate tumor progression and therapeutic response. In turn, anticancer treatments may alter the composition and functions of the TME. To investigate this continuous dialog in the context of primary brain cancers, we developed a multimodal longitudinal imaging strategy. We combined macroscopical magnetic resonance imaging with subcellular resolution two-photon intravital microscopy, and leveraged the power of single-cell analysis tools to gain insights into the ongoing interactions between different components of the TME and cancer cells. Our experiments revealed that the migratory behavior of tumor-associated macrophages is different in genetically distinct glioblastomas, and in response to macrophage-targeted therapy. These results underscore the importance of studying cancer longitudinally in an in vivo setting, to reveal complex and dynamic alterations in the TME during disease progression and therapeutic intervention.

During puberty, the mouse mammary gland develops into a highly branched epithelial network. Owing to the absence of exclusive stem cell markers, the location, multiplicity, dynamics and fate of mammary stem cells (MaSCs), which drive branching morphogenesis, are unknown. Here we show that morphogenesis is driven by proliferative terminal end buds that terminate or bifurcate witwh near equal probability, in a stochastic and time-invariant manner, leading to a heterogeneous epithelial network. We show that the majority of terminal end bud cells function as highly proliferative, lineage-committed MaSCs that are heterogeneous in their expression profile and short-term contribution to ductal extension. Yet, through cell rearrangements during terminal end bud bifurcation, each MaSC is able to contribute actively to long-term growth. Our study shows that the behaviour of MaSCs is not directly linked to a single expression profile. Instead, morphogenesis relies upon lineage-restricted heterogeneous MaSC populations that function as single equipotent pools in the long term.

Introduction Tumours consist of heterogeneous populations of cancer cells with various abilities to metastasize. Recent data shows that cancer cells from different subtypes exchange biomolecules through extracellular vesicles (EVs), influencing each other’s metastatic behaviour. How EVs can mediate this effect is still largely unknown, especially in tumours from the same subtype in which molecular differences are small but instrumental drivers of metastasis. Material and methods Here, we study EVs shed by two B16 melanoma lines with different metastatic potential. Using label-free tandem mass spectrometry and RNA sequencing we profile the tumour microenvironmental EVs shed by these clones in vivo. We use the Cre-LoxP system to monitor EV transfer in vivo in combination with intravital microscopy to study phenotypic changes induced by EV exchange. Results and discussions We show that melanoma cells with different metastatic capacities functionally exchange EVs in vivo. EVs shed into the tumour microenvironment by cancer cells contain interconnected protein and RNA signalling networks involved in a variety of processes, including cell migration. Transfer of EVs from highly metastatic and migratory cells copies their phenotype to other cancer cells. Conclusion We show that cancer cells influence each other’s behaviour in the tumour microenvironment through EVs. Profiling of tumor-derived EVs suggests that this is mediated by a diverse range of EV-molecules that together amplify numerous nodes of signalling networks in recipient cells.

The rapid turnover of the mammalian intestinal epithelium is supported by stem cells located around the base of the crypt. In addition to the Lgr5 marker, intestinal stem cells have been associated with other markers that are expressed heterogeneously within the crypt base region. Previous quantitative clonal fate analyses have led to the proposal that homeostasis occurs as the consequence of neutral competition between dividing stem cells. However, the short-term behaviour of individual Lgr5(+) cells positioned at different locations within the crypt base compartment has not been resolved. Here we establish the short-term dynamics of intestinal stem cells using the novel approach of continuous intravital imaging of Lgr5- Confetti mice. We find that Lgr5(+) cells in the upper part of the niche (termed 'border cells') can be passively displaced into the transit-amplifying domain, after the division of proximate cells, implying that the determination of stem-cell fate can be uncoupled from division. Through quantitative analysis of individual clonal lineages, we show that stem cells at the crypt base, termed 'central cells', experience a survival advantage over border stem cells. However, through the transfer of stem cells between the border and central regions, all Lgr5(+) cells are endowed with long-term self-renewal potential. These findings establish a novel paradigm for stem-cell maintenance in which a dynamically heterogeneous cell population is able to function long term as a single stem-cell pool.

Breast cancer is the second cause of death in Europe with an incidence of 464.00 new cases in 2012. Chemotherapy is frequently used to treat severe breast cancer. Current drugs like taxanes and anthracycline used alone or in combination are efficient. Moreover the use of biomarkers, like HER2 and BRCA status is now helping in the choice of the most adapted chemotherapy to the patient. De novo or acquired resistance limits, however, the clinical usefulness of drugs used in current chemotherapy. The development of new therapeutics effective against drug-resistant cancers thus still represents an important challenge. Our team has discovered a new LIM Kinases (LIMK1/2) inhibitor, “LIM-Pyr1”. LIMK1/2 are situated at a crossroads of several signaling pathways mainly activated by tyrosine kinase receptors and regulate both actin and microtubules dynamics. LIMK1/2 regulate actin dynamics through cofilin phosphorylation. Cofilin is an actin-depolymerizing factor and its phosphorylation inactivates its actin severing activity. LIMK1/2 also regulate microtubule dynamics through a mechanism unknown yet. LIMK1/2 inhibition induce microtubules stabilization (1). Moreover, LIMK1/2 are overexpressed in many invasive cancers and appear to be a relevant target for anticancer therapy (2, 3). We have shown that LIM-Pyr1 is toxic on cell lines resistant to conventional chemotherapy (4) and tested LIM-Pyr1 therapeutic activity on different breast cancer models (xenografts in mice). We found that LIM-Pyr1 shows a potent antitumor activity both on primary and secondary tumors, with no detectable undesirable side effects. The antitumor effect is effective on paclitaxel resistant xenografts. Finally, intravital microscopy analysis indicates that a LIM-Pyr1 treatment induces a strong morphological change of tumor cells inside the tumors and reduces their migration. LIM-Pyr1 and its derivatives could thus represent a pharmacological alternative to overcome resistances often observed when tumors are treated with microtubule targeting agents. (1) O. Bernard, LIM Kinases, regulators of actin dynamics, Int. Journals of Biochemistry and cell biology (2007) 1071-1076 (2) F. Manetti, Recent finding confirm LIM Domain Kinases as emerging target candidates for cancer therapy, Curr. Cancer Drug Targets (2012) 12,543-560 (3) W. Wang, R. Eddy, J. Condeelis, The cofilin pathway in breast cancer invasion and metastasis, Nat. Cancer Reviews (2007) (4) R. Prudent, E. Vassal-Stermann, C-H Nguyen et al., Pharmacological inhibition of LIM Kinases stabilizes microtubules, Cancer Research (2012) Citation Format: Chloé Prunier, Julien Vollaire, Véronique Josserand, Anoek Zomer, Amandine Hurbin, Renaud Prudent, Pascale Cohen, Jacco van Rheenen, Jean-Luc Coll, Marc Billaud, Laurence Lafanechère. Anti-cancer activity of a new LIM-Kinases inhibitor: “LIM-Pyr1”. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5399. doi:10.1158/1538-7445.AM2015-5399

Interleukin (IL)-10 is a major cancer-related immunosuppressive factor, exhibiting a unique ability to hamper the maturation of dendritic cells (DCs). We have previously reported that IL-10 induces the conversion of activated, migratory CD1a+ DCs found in the human skin to CD14+CD141+ macrophage-like cells. Here, as a model of tumor-conditioned DC maturation, we functionally assessed CD14- and CD14+ DCs that matured in vitro upon exposure to IL-10. IL-10-induced CD14+ DCs were phenotypically characterized by a low maturation state as well as by high levels of BDCA3 and DC-SIGN, and as such they closely resembled CD14+ cells infiltrating melanoma metastases. Compared with DC matured under standard conditions, CD14+ DCs were found to express high levels of B7-H1 on the cell surface, to secrete low levels of IL-12p70, to preferentially induce TH2 cells, to have a lower allogeneic TH cell and tumor antigen-specific CD8+ T-cell priming capacity and to induce proliferative T-cell anergy. In contrast to their CD14+ counterparts, CD14- monocyte-derived DCs retained allogeneic TH priming capacity but induced a functionally anergic state as they completely abolished the release of effector cytokines. Transcriptional and cytokine release profiling studies indicated a more profound angiogenic and pro-invasive signature of CD14+ DCs as compared with DCs matured in standard conditions or CD14− DCs matured in the presence of IL-10. Importantly, signal transducer and activator of transcription 3 (STAT3) depletion by RNA interference prevented the development of the IL-10-associated CD14+ phenotype, allowing for normal DC maturation and providing a potential means of therapeutic intervention.