CD4+ T cell-induced inflammatory cell death controls immune-evasive tumours.

Most clinically applied cancer immunotherapies rely on the ability of CD8+ cytolytic T cells to directly recognize and kill tumour cells1-3. These strategies are limited by the emergence of major histocompatibility complex (MHC)-deficient tumour cells and the formation of an immunosuppressive tumour microenvironment4-6. The ability of CD4+ effector cells to contribute to antitumour immunity independently of CD8+ T cells is increasingly recognized, but strategies to unleash their full potential remain to be identified7-10. Here, we describe a mechanism whereby a small number of CD4+ T cells is sufficient to eradicate MHC-deficient tumours that escape direct CD8+ T cell targeting. The CD4+ effector T cells preferentially cluster at tumour invasive margins where they interact with MHC-II+CD11c+ antigen-presenting cells. We show that T helper type 1 cell-directed CD4+ T cells and innate immune stimulation reprogramme the tumour-associated myeloid cell network towards interferon-activated antigen-presenting and iNOS-expressing tumouricidal effector phenotypes. Together, CD4+ T cells and tumouricidal myeloid cells orchestrate the induction of remote inflammatory cell death that indirectly eradicates interferon-unresponsive and MHC-deficient tumours. These results warrant the clinical exploitation of this ability of CD4+ T cells and innate immune stimulators in a strategy to complement the direct cytolytic activity of CD8+ T cells and natural killer cells and advance cancer immunotherapies.

Single-cell molecular profiling using ex vivo functional readouts fuels precision oncology in glioblastoma.

BACKGROUND: Functional profiling of freshly isolated glioblastoma (GBM) cells is being evaluated as a next-generation method for precision oncology. While promising, its success largely depends on the method to evaluate treatment activity which requires sufficient resolution and specificity. METHODS: Here, we describe the 'precision oncology by single-cell profiling using ex vivo readouts of functionality' (PROSPERO) assay to evaluate the intrinsic susceptibility of high-grade brain tumor cells to respond to therapy. Different from other assays, PROSPERO extends beyond life/death screening by rapidly evaluating acute molecular drug responses at single-cell resolution. RESULTS: The PROSPERO assay was developed by correlating short-term single-cell molecular signatures using mass cytometry by time-of-flight (CyTOF) to long-term cytotoxicity readouts in representative patient-derived glioblastoma cell cultures (n = 14) that were exposed to radiotherapy and the small-molecule p53/MDM2 inhibitor AMG232. The predictive model was subsequently projected to evaluate drug activity in freshly resected GBM samples from patients (n = 34). Here, PROSPERO revealed an overall limited capacity of tumor cells to respond to therapy, as reflected by the inability to induce key molecular markers upon ex vivo treatment exposure, while retaining proliferative capacity, insights that were validated in patient-derived xenograft (PDX) models. This approach also allowed the investigation of cellular plasticity, which in PDCLs highlighted therapy-induced proneural-to-mesenchymal (PMT) transitions, while in patients' samples this was more heterogeneous. CONCLUSION: PROSPERO provides a precise way to evaluate therapy efficacy by measuring molecular drug responses using specific biomarker changes in freshly resected brain tumor samples, in addition to providing key functional insights in cellular behavior, which may ultimately complement standard, clinical biomarker evaluations.

GSNOR deficiency promotes tumor growth via FAK1 S-nitrosylation.

Nitric oxide (NO) production in the tumor microenvironment is a common element in cancer. S-nitrosylation, the post-translational modification of cysteines by NO, is emerging as a key transduction mechanism sustaining tumorigenesis. However, most oncoproteins that are regulated by S-nitrosylation are still unknown. Here we show that S-nitrosoglutathione reductase (GSNOR), the enzyme that deactivates S-nitrosylation, is hypo-expressed in several human malignancies. Using multiple tumor models, we demonstrate that GSNOR deficiency induces S-nitrosylation of focal adhesion kinase 1 (FAK1) at C658. This event enhances FAK1 autophosphorylation and sustains tumorigenicity by providing cancer cells with the ability to survive in suspension (evade anoikis). In line with these results, GSNOR-deficient tumor models are highly susceptible to treatment with FAK1 inhibitors. Altogether, our findings advance our understanding of the oncogenic role of S-nitrosylation, define GSNOR as a tumor suppressor, and point to GSNOR hypo-expression as a therapeutically exploitable vulnerability in cancer.

Mapping the Immune Landscape in Metastatic Melanoma Reveals Localized Cell-Cell Interactions That Predict Immunotherapy Response.

While immune checkpoint-based immunotherapy (ICI) shows promising clinical results in patients with cancer, only a subset of patients responds favorably. Response to ICI is dictated by complex networks of cellular interactions between malignant and nonmalignant cells. Although insights into the mechanisms that modulate the pivotal antitumoral activity of cytotoxic T cells (Tcy) have recently been gained, much of what has been learned is based on single-cell analyses of dissociated tumor samples, resulting in a lack of critical information about the spatial distribution of relevant cell types. Here, we used multiplexed IHC to spatially characterize the immune landscape of metastatic melanoma from responders and nonresponders to ICI. Such high-dimensional pathology maps showed that Tcy gradually evolve toward an exhausted phenotype as they approach and infiltrate the tumor. Moreover, a key cellular interaction network functionally linked Tcy and PD-L1+ macrophages. Mapping the respective spatial distributions of these two cell populations predicted response to anti-PD-1 immunotherapy with high confidence. These results suggest that baseline measurements of the spatial context should be integrated in the design of predictive biomarkers to identify patients likely to benefit from ICI. SIGNIFICANCE: This study shows that spatial characterization can address the challenge of finding efficient biomarkers, revealing that localization of macrophages and T cells in melanoma predicts patient response to ICI. See related commentary by Smalley and Smalley, p. 3198.

Activation of the integrated stress response confers vulnerability to mitoribosome-targeting antibiotics in melanoma.

The ability to adapt to environmental stress, including therapeutic insult, contributes to tumor evolution and drug resistance. In suboptimal conditions, the integrated stress response (ISR) promotes survival by dampening cytosolic translation. We show that ISR-dependent survival also relies on a concomitant up-regulation of mitochondrial protein synthesis, a vulnerability that can be exploited using mitoribosome-targeting antibiotics. Accordingly, such agents sensitized to MAPK inhibition, thus preventing the development of resistance in BRAFV600E melanoma models. Additionally, this treatment compromised the growth of melanomas that exhibited elevated ISR activity and resistance to both immunotherapy and targeted therapy. In keeping with this, pharmacological inactivation of ISR, or silencing of ATF4, rescued the antitumoral response to the tetracyclines. Moreover, a melanoma patient exposed to doxycycline experienced complete and long-lasting response of a treatment-resistant lesion. Our study indicates that the repurposing of mitoribosome-targeting antibiotics offers a rational salvage strategy for targeted therapy in BRAF mutant melanoma and a therapeutic option for NRAS-driven and immunotherapy-resistant tumors.

Loss of Ambra1 promotes melanoma growth and invasion.

Melanoma is the deadliest skin cancer. Despite improvements in the understanding of the molecular mechanisms underlying melanoma biology and in defining new curative strategies, the therapeutic needs for this disease have not yet been fulfilled. Herein, we provide evidence that the Activating Molecule in Beclin-1-Regulated Autophagy (Ambra1) contributes to melanoma development. Indeed, we show that Ambra1 deficiency confers accelerated tumor growth and decreased overall survival in Braf/Pten-mutated mouse models of melanoma. Also, we demonstrate that Ambra1 deletion promotes melanoma aggressiveness and metastasis by increasing cell motility/invasion and activating an EMT-like process. Moreover, we show that Ambra1 deficiency in melanoma impacts extracellular matrix remodeling and induces hyperactivation of the focal adhesion kinase 1 (FAK1) signaling, whose inhibition is able to reduce cell invasion and melanoma growth. Overall, our findings identify a function for AMBRA1 as tumor suppressor in melanoma, proposing FAK1 inhibition as a therapeutic strategy for AMBRA1 low-expressing melanoma.

BNIP3 promotes HIF-1α-driven melanoma growth by curbing intracellular iron homeostasis.

BNIP3 is a mitophagy receptor with context-dependent roles in cancer, but whether and how it modulates melanoma growth in vivo remains unknown. Here, we found that elevated BNIP3 levels correlated with poorer melanoma patient's survival and depletion of BNIP3 in B16-F10 melanoma cells compromised tumor growth in vivo. BNIP3 depletion halted mitophagy and enforced a PHD2-mediated downregulation of HIF-1α and its glycolytic program both in vitro and in vivo. Mechanistically, we found that BNIP3-deprived melanoma cells displayed increased intracellular iron levels caused by heightened NCOA4-mediated ferritinophagy, which fostered PHD2-mediated HIF-1α destabilization. These effects were not phenocopied by ATG5 or NIX silencing. Restoring HIF-1α levels in BNIP3-depleted melanoma cells rescued their metabolic phenotype and tumor growth in vivo, but did not affect NCOA4 turnover, underscoring that these BNIP3 effects are not secondary to HIF-1α. These results unravel an unexpected role of BNIP3 as upstream regulator of the pro-tumorigenic HIF-1α glycolytic program in melanoma cells.

Rethinking the biology of metastatic melanoma: a holistic approach.

Over the past decades, melanoma-related mortality has remained nearly stable. The main reason is treatment failure of metastatic disease and the inherently linked knowledge gap regarding metastasis formation. In order to elicit invasion, melanoma cells manipulate the tumor microenvironment, gain motility, and adhere to the extracellular matrix and cancer-associated fibroblasts. Melanoma cells thereby express different cell adhesion molecules like laminins, integrins, N-cadherin, and others. Epithelial-mesenchymal transition (EMT) is physiological during embryologic development, but reactivated during malignancy. Despite not being truly epithelial, neural crest-derived malignancies like melanoma share similar biological programs that enable tumorigenesis, invasion, and metastasis. This complex phenomenon is termed phenotype switching and is intertwined with oncometabolism as well as dormancy escape. Additionally, it has been shown that primary melanoma shed exosomes that create a favorable premetastatic niche in the microenvironment of secondary organs and lymph nodes. Although the growing body of literature describes the aforementioned concepts separately, an integrative holistic approach is missing. Using melanoma as a tumor model, this review will shed light on these complex biological principles in an attempt to clarify the mechanistic metastatic pathways that dictate tumor and patient fate.

Antibodies validated for routinely processed tissues stain frozen sections unpredictably.

Background: Antibody validation for tissue staining is required for reproducibility; criteria to ensure validity have been published recently. The majority of these recommendations imply the use of routinely processed (formalin-fixed, paraffin-embedded) tissue. Materials & methods: We applied to lightly fixed frozen sections a panel of 126 antibodies validated for formalin-fixed, paraffin-embedded tissue with extended criteria. Results: Less than 30% of the antibodies performed as expected with all fixations. 35% preferred one fixation over another, 13% gave nonspecific staining and 23% did not stain at all. Conclusion: Individual antibody variability of the paratope's fitness for the fixed antigen may be the cause. Revalidation of established antibody panels is required when they are applied to sections whose fixation and processing are different from the tissue where they were initially validated.

Integrative histopathological and immunophenotypical characterisation of the inflammatory microenvironment in spitzoid melanocytic neoplasms.

AIMS: The role of inflammation in conventional cutaneous melanoma has been extensively studied, whereas only little is known about the inflammatory microenvironment and immunogenic properties of spitzoid melanocytic neoplasms. The composition of infiltrating immune cells and the architectural distribution of the inflammation, in particular, are still obscure. This is the first study, to our knowledge, to systematically characterise the inflammatory patterns and the leucocyte subsets in spitzoid melanocytic lesions. METHODS AND RESULTS: We examined 79 spitzoid neoplasms including banal Spitz naevi (SN, n = 50), atypical Spitz tumours (AST, n = 17) and malignant Spitz tumours (MST, n = 12) using histopathological analysis and immunohistochemistry. Spitzoid melanocytic lesions showed a high frequency (67.1%, n = 53 of 79) of inflammation. Four inflammatory patterns were identified according to architectural composition, distribution and intensity of inflammation. The majority of the inflammatory infiltrate corresponded to CD3+ /CD8+ T lymphocytes (56.1%), followed by CD3+ /CD4+ T cells (35.7%) and CD68+ histiocytes (20.3%). CD3+ /TIA-1+ cytotoxic T lymphocytes constituted 3.7% of inflammatory cells. Rarely, CD3+ / granzyme B+ cytotoxic T lymphocytes (2.7%) and CD138+ plasma cells (0.5%) were detected in the infiltrating immune cells. There was no significant difference in the inflammatory cellular composition among the spitzoid melanocytic subgroups (SN versus AST versus MST). CONCLUSION: Our findings demonstrate that Spitz tumours are highly immunogenic lesions. Inflammation with the presence of lymphocytic aggregates predominated in SN, but was not distinctive for this melanocytic category. A strong and intense inflammation was suggestive of an underlying malignancy. The infiltrating cytotoxic T lymphocyte subsets in Spitz tumours deserve further investigation in larger study cohorts to elucidate prognostic and immuno-oncological therapeutic relevance.

The Adaptive and Innate Immune Cell Landscape of Uterine Leiomyosarcomas.

Reactivation of the anti-tumor response has shown substantial progress in aggressive tumors such as melanoma and lung cancer. Data on less common histotypes are scanty. Immune checkpoint inhibitor therapy has been applied to few cases of uterine leiomyosarcomas, of which the immune cell composition was not examined in detail. We analyzed the inflammatory infiltrate of 21 such cases in high-dimensional, single cell phenotyping on routinely processed tissue. T-lymphoid cells displayed a composite phenotype common to all tumors, suggestive of antigen-exposure, acute and chronic exhaustion. To the contrary, myelomonocytic cells had case-specific individual combinations of phenotypes and subsets. We identified five distinct monocyte-macrophage cell types, some not described before, bearing immunosuppressive molecules (TIM3, B7H3, VISTA, PD1, PDL1). Detailed in situ analysis of routinely processed tissue yields comprehensive information about the immune status of sarcomas. The method employed provides equivalent information to extractive single-cell technology, with spatial contexture and a modest investment.

Plasma cells in primary melanoma. Prognostic significance and possible role of IgA.

Melanoma is not only one of the most immunogenic cancers but also one of the most effective cancers at subverting host immunity. The role of T lymphocytes in tumor immunity has been extensively studied in melanoma, whereas less is known about the importance of B lymphocytes. The effects of plasma cells (PCs), in particular, are still obscure. The aim of this study was to characterize pathological features and clinical outcome of primary cutaneous melanomas associated with PCs. Moreover, we investigated the origins of the melanoma-associated PCs. Finally, we studied the outcome of patients with primary melanomas with PCs. We reviewed 710 melanomas to correlate the presence of PCs with histological prognostic markers. Immunohistochemistry for CD138 and heavy and light chains was performed in primary melanomas (PM) and in loco-regional lymph nodes (LN), both metastatic and not metastatic. In three PM and nine LN with frozen material, VDJ-rearrangement was analyzed by Gene Scan Analysis. Survival analysis was performed on a group of 85 primary melanomas >2 mm in thickness. Forty-one cases (3.7%) showed clusters/sheets of PCs. PC-rich melanomas occurred at an older age and were thicker, more often ulcerated and more mitotically active (P<0.05). PCs were polyclonal and often expressed IgA in addition to IgG. In LN, clusters/sheets of IgA+ PCs were found both in the sinuses and subcapsular areas. Analysis of VDJ-rearrangements showed the IgA to be oligoclonal. Melanomas with clusters/sheets of PCs had a significantly worse survival compared with melanomas without PCs while, interestingly, melanomas with sparse PCs were associated with a better clinical outcome (P=0.002). In conclusion, melanomas with sheets/clusters of PCs are associated with worse prognosis. IgG and IgA are the isotypes predominantly produced by these PCs. IgA oligoclonality suggests an antigen-driven response that facilitates melanoma progression by a hitherto unknown mechanism.

Expression of T-follicular helper markers in sequential biopsies of progressive mycosis fungoides and other primary cutaneous T-cell lymphomas.

T-follicular helper (Tfh) lymphocytes represent the neoplastic cells of angioimmunoblastic T-cell lymphoma and have been observed also in several cutaneous T-cell lymphomas (CTCLs) and extracutaneous T-cell lymphomas, including peripheral T-cell lymphoma, not otherwise specified, mycosis fungoides (MF), cutaneous CD4 small/medium T-cell lymphoma (CD4SMTCL), and Sezary syndrome. We studied a large number of different types of primary CTCL for expression of Tfh markers, including 36 biopsies from 21 patients with MF (with sequential biopsies from patch stage and tumor stage of 15 patients), 13 patients with CD4SMTCL, 9 with lymphomatoid papulosis, 11 with cutaneous anaplastic large cell lymphoma (cALCL), 2 with cutaneous γ/δ T-cell lymphoma, 8 with subcutaneous panniculitis-like T-cell lymphoma, 3 with cutaneous aggressive epidermotropic CD8 cytotoxic T-cell lymphoma, 6 with cutaneous peripheral T-cell lymphoma, not otherwise specified, and 1 with Sezary syndrome. Expression of at least 3 of 5 markers (PD-1, CXCL-13, ICOS, Bcl-6, and CD10) in >10% of tumor cells was observed in 33 biopsies (MF = 20; CD4SMTCL = 11; 1 each cutaneous anaplastic large cell lymphoma and subcutaneous panniculitis-like T-cell lymphoma, respectively). Our study shows that a Tfh phenotype is very common in MF and CD4SMTCL but can be observed rarely also in other types of CTCL. Expression of Tfh markers should not be used for classification of any entity of CTCL and may only integrate other immunohistochemical stainings for a more accurate characterization of these disorders. Precise distinction of Tfh-positive CTCLs from secondary skin manifestations of angioimmunoblastic T-cell lymphoma cannot rest on demonstration of a Tfh phenotype alone and should be achieved by a synthesis of clinical, histological, and phenotypic features.

Tissue microarray design and construction for scientific, industrial and diagnostic use.

CONTEXT: In 2013 the high throughput technology known as Tissue Micro Array (TMA) will be fifteen years old. Its elements (design, construction and analysis) are intuitive and the core histopathology technique is unsophisticated, which may be a reason why has eluded a rigorous scientific scrutiny. The source of errors, particularly in specimen identification and how to control for it is unreported. Formal validation of the accuracy of segmenting (also known as de-arraying) hundreds of samples, pairing with the sample data is lacking. AIMS: We wanted to address these issues in order to bring the technique to recognized standards of quality in TMA use for research, diagnostics and industrial purposes. RESULTS: We systematically addressed the sources of error and used barcode-driven data input throughout the whole process including matching the design with a TMA virtual image and segmenting that image back to individual cases, together with the associated data. In addition we demonstrate on mathematical grounds that a TMA design, when superimposed onto the corresponding whole slide image, validates on each and every sample the correspondence between the image and patient's data. CONCLUSIONS: High throughput use of the TMA technology is a safe and efficient method for research, diagnosis and industrial use if all sources of errors are identified and addressed.