Cell growth in aggregates determines gene expression, proliferation, survival, chemoresistance, and sensitivity to immune effectors in follicular lymphoma.

Lymphomas grow as dense aggregates in patients, but whether this spatial organization affects lymphoma cell biology is unknown. We grew follicular lymphoma (FL) cells in vitro as multicellular aggregates of lymphoma cells to investigate this question. Gene expression analysis revealed that 612 genes were differentially expressed when cells grew in multicellular aggregates of lymphoma cells rather than in suspension. These genes correspond to several GO biological processes, such as hypoxia, activation of NF-κB pathway, and negative regulation of cell cycle, a gene signature also found in the transcriptomes from FL biopsies. Pimonidazole staining, HIF-1A accumulation, and VEGFA release confirmed that cells in multicellular aggregates of lymphoma cells actually respond to hypoxia. In adaptation to such conditions, they also displayed an activated NF-κB pathway and a quiescent status far more frequently than in suspension. When cultured in three dimensions, FL cells display resistance to doxorubicin and bendamustine, two drugs largely used in FL therapy, compared to FL cultured in suspension. Finally, multicellular aggregates of lymphoma cells were also found to be less sensitive to purified natural killer cells. To conclude, our study shows that in FL, spatial organization results in dramatic changes in FL biology, including gene expression, proliferation, drug resistance, and immune escape.

Endogenous IL-8 acts as a CD16 co-activator for natural killer-mediated anti-CD20 B cell depletion in chronic lymphocytic leukemia.

Rituximab (RTX, anti-CD20 antibody) combined with chemotherapy is currently standard treatment for chronic lymphocytic leukemia (CLL). Serum level of IL-8 is a prognostic factor for CLL that correlates with disease stage. We investigated whether endogenous IL-8 affects RTX or Obinutuzumab (GA101) B-leukemic depletion mediated by natural killers (NK). Using whole peripheral blood lymphocytes from untreated CLL patients, RTX, but most significantly GA101, were effective in B-cell depletion and NK activation. IL-8 inhibition completely inhibited B-cell depletion by RTX and reduced GA101-induced B-cell depletion. Altogether results underline IL-8 as an endogenous NK co-activator and confirm GA101 therapeutic potential for CLL treatment.

What lessons can be learned from γδ T cell-based cancer immunotherapy trials?

During the last several years, research has produced a significant amount of knowledge concerning the characteristics of human γδ T lymphocytes. Findings regarding the immune functions of these cells, particularly their natural killer cell-like lytic activity against tumor cells, have raised expectations for the therapeutic applications of these cells for cancer. Pharmaceutical companies have produced selective agonists for these lymphocytes, and several teams have launched clinical trials of γδ T cell-based cancer therapies. The findings from these studies include hematological malignancies (follicular lymphoma, multiple myeloma, acute and chronic myeloid leukemia), as well as solid tumors (renal cell, breast and prostate carcinomas), consisting of samples from more than 250 patients from Europe, Japan and the United States. The results of these pioneering studies are now available, and this short review summarizes the lessons learned and the role of γδ T cell-based strategies in the current landscape of cancer immunotherapies.

Epidermal growth factor receptor/beta-catenin/T-cell factor 4/matrix metalloproteinase 1: a new pathway for regulating keratinocyte invasiveness after UVA irradiation.

Previous studies have established that UV irradiation results in epidermal growth factor receptor (EGFR) activation in keratinocytes. However, the signaling pathways and cellular effects related to this process remain incompletely elucidated. Herein, we describe for the first time that UVA-mediated EGFR activation results in beta-catenin tyrosine phosphorylation at the Y654 residue responsible for the dissociation of E-cadherin/alpha-catenin/beta-catenin complexes. Moreover, UVA induces an EGFR-dependent, but Wnt-independent, beta-catenin relocalization from the membrane to the nucleus followed by its association with T-cell factor 4 (TCF4). This newly formed beta-catenin/TCF4 complex binds to a specific site on matrix metalloproteinase 1 (MMP1) promoter and governs MMP1 gene and protein expression, as well as cell migration in collagen and gelatin. Altogether, these results suggest that UVA stimulates keratinocyte invasiveness through two coordinated EGFR-dependent processes: loss of cell-to-cell contact due to beta-catenin/E-cadherin/alpha-catenin dissociation and increased cell migration through extracellular matrix component degradation due to beta-catenin/TCF4-dependent MMP1 regulation. These events may represent an important step in epidermis repair following UVA injury and their abnormal regulation could contribute to photoaging and photocarcinogenesis.

PKC zeta mTOR pathway: a new target for rituximab therapy in follicular lymphoma.

Previous studies have documented that, in malignant B cells, rituximab elicits a complex and not yet totally understood signaling network contributing to its antitumor effect. In this context, we investigated the role of protein kinase C zeta (PKCzeta), an atypical PKC isoform, in the cellular response to rituximab. We found that follicular lymphoma cells displayed an increase in PKCzeta expression and activity levels, compared with nonmalignant B cells, and that this enzyme was a critical regulator of the classical MAPK module by stimulating Raf-1 kinase activity. PKCzeta appeared to be a significant contributor of abnormal mTOR regulation in follicular lymphoma cells through a MAPK-dependent mechanism. Rituximab was found to inhibit the PKCzeta/MAPK/mTOR module in these cells but not in other B-cell lymphomas. Importantly, the expression of a constitutively active form of PKCzeta resulted in an efficient protection of these cells toward rituximab. Altogether, our study describes a new regulatory component of mTOR pathway in follicular cell lymphoma and demonstrates that PKCzeta is a target for rituximab. Therefore, PKCzeta could represent an important parameter for rituximab efficacy and a promising target for future targeted therapy in follicular lymphoma.

Epidermal growth factor receptor pathway mitigates UVA-induced G2/M arrest in keratinocyte cells.

UVA irradiation contributes largely to photocarcinogenesis. In the process of keratinocyte transformation, the activation of EGFR by UV is now considered as a critical event. However, the mechanism that links the EGFR pathway and photocarcinogenesis is not totally understood. In this study, we report that the EGFR/Akt pathway mitigated G2/M arrest in human HaCaT keratinocytes and normal human keratinocytes treated with low doses of UVA irradiation. EGFR-mediated Akt activation resulted in increased level of checkpoint 1 kinase (Chk1) inhibitory phosphorylation (Ser280). In contrast, EGFR/Akt pathway inhibition resulted in the abrogation of Ser280 Chk1 phosphorylation, increased level of Chk1 stimulatory phosphorylation (Ser345), and restoration of G2/M arrest. Altogether, these results suggest that, after UVA exposure, the EGFR/Akt pathway subverts the G2/M checkpoint. This effect may have serious implications in photocarcinogenesis by allowing damaged cells to transit through the cell cycle.

Statistical behavior of joint least-square estimation in the phase diversity context.

The images recorded by optical telescopes are often degraded by aberrations that induce phase variations in the pupil plane. Several wavefront sensing techniques have been proposed to estimate aberrated phases. One of them is phase diversity, for which the joint least-square approach introduced by Gonsalves et al. is a reference method to estimate phase coefficients from the recorded images. In this paper, we rely on the asymptotic theory of Toeplitz matrices to show that Gonsalves' technique provides a consistent phase estimator as the size of the images grows. No comparable result is yielded by the classical joint maximum likelihood interpretation (e.g., as found in the work by Paxman et al.). Finally, our theoretical analysis is illustrated through simulated problems.

Marginal estimation of aberrations and image restoration by use of phase diversity.

We propose a novel method called marginal estimator for estimating the aberrations and the object from phase-diversity data. The conventional estimator found in the literature concerning the technique first proposed by Gonsalves has its basis in a joint estimation of the aberrated phase and the observed object. By means of simulations, we study the behavior of the conventional estimator, which is interpretable as a joint maximum a posteriori approach, and we show in particular that it has undesirable asymptotic properties and does not permit an optimal joint estimation of the object and the aberrated phase. We propose a novel marginal estimator of the sole phase by maximum a posteriori. It is obtained by integrating the observed object out of the problem. This reduces drastically the number of unknowns, allows the unsupervised estimation of the regularization parameters, and provides better asymptotic properties. We show that the marginal method is also appropriate for the restoration of the object. This estimator is implemented and its properties are validated by simulations. The performance of the joint method and the marginal one is compared on both simulated and experimental data in the case of Earth observation. For the studied object, the comparison of the quality of the phase restoration shows that the performance of the marginal approach is better under high-noise-level conditions.