Prevention of Prostate Cancer Metastasis by a CRISPR-delivering Nanoplatform for Interleukin-30 Genome Editing.

Prostate-cancer (PC) is a leading cause of cancer-related deaths in men worldwide. Interleukin-(IL)-30 is a PC-progression driver, and its suppression would be strategic for fighting metastatic disease. Biocompatible Lipid-Nanoparticles (NPs) were loaded with CRISPR/Cas9gRNA to delete human(h)IL30-gene and functionalized with anti-PSCA-Abs (Cas9hIL30-PSCA-NPs). Efficiency of the NPs in targeting IL30 and metastatic potential of PC cells was examined in vivo, in xenograft models of lung metastasis, and in vitro, by using 2-Organ-on-Chip (2-OC), containing 3D-spheroids of IL30+PC-Endothelial-Cell(EC) co-cultures in circuit with either Lung-mimicking-spheroids, or Bone-marrow(BM)-niche-mimicking-scaffolds. Cas9hIL30-PSCA-NPs demonstrated circulation stability, genome editing efficiency, without off-target effects and organ toxicity. Intravenous injection of three-doses/13-days, or five-doses/20-days, of NPs in mice bearing circulating PC cells and micro-emboli substantially hindered lung metastasization. Cas9hIL30-PSCA-NPs inhibited PC cell proliferation and expression of IL30 and metastasis-drivers, such as CXCR2, CXCR4, IGF1, L1CAM, METAP2, MMP2 and TNFSF10, whereas CDH1 was up-regulated. PC-Lung and PC-BM 2-OCs revealed that Cas9hIL30-PSCA-NPs suppressed PC cell release of CXCL2/GROß, which in vivo was associated with intra-metastatic myeloid cell infiltrates, and of DKK1, OPG and IL6, which in vitro boosted endothelial-network formation and cancer cell migration. Development of a patient-tailored nanoplatform for selective CRISPR-mediated IL30 gene deletion is a clinically valuable tool against PC progression.

The multifaceted role of the stroma in the healthy prostate and prostate cancer.

Prostate cancer (PC) is an age-related disease and represents, after lung cancer, the second cause of cancer death in males worldwide. Mortality is due to the metastatic disease, which mainly involves the bones, lungs, and liver. In the last 20 years, the incidence of metastatic PC has increased in Western Countries, and a further increase is expected in the near future, due to the population ageing. Current treatment options, including state of the art cancer immunotherapy, need to be more effective to achieve long-term disease control. The most significant anatomical barrier to overcome to improve the effectiveness of current and newly designed drug strategies consists of the prostatic stroma, in particular the fibroblasts and the extracellular matrix, which are the most abundant components of both the normal and tumor prostatic microenvironment. By weaving a complex communication network with the glandular epithelium, the immune cells, the microbiota, the endothelium, and the nerves, in the healthy prostatic microenvironment, the fibroblasts and the extracellular matrix support organ development and homeostasis. However, during inflammation, ageing and prostate tumorigenesis, they undergo dramatic phenotypic and genotypic changes, which impact on tumor growth and progression and on the development of therapy resistance. Here, we focus on the characteristics and functions of the prostate associated fibroblasts and of the extracellular matrix in health and cancer. We emphasize their roles in shaping tumor behavior and the feasibility of manipulating and/or targeting these stromal components to overcome the limitations of current treatments and to improve precision medicine's chances of success.

Oxidative Stress, Mutations and Chromosomal Aberrations Induced by In Vitro and In Vivo Exposure to Furan.

Furan is a volatile compound that is formed in foods during thermal processing. It is classified as a possible human carcinogen by international authorities based on sufficient evidence of carcinogenicity from studies in experimental animals. Although a vast number of studies both in vitro and in vivo have been performed to investigate furan genotoxicity, the results are inconsistent, and its carcinogenic mode of action remains to be clarified. Here, we address the mutagenic and clastogenic activity of furan and its prime reactive metabolite cis-2 butene-1,4-dial (BDA) in mammalian cells in culture and in mouse animal models in a search for DNA lesions responsible of these effects. To this aim, Fanconi anemia-derived human cell lines defective in the repair of DNA inter-strand crosslinks (ICLs) and Ogg1-/- mice defective in the removal of 8-hydroxyguanine from DNA, were used. We show that both furan and BDA present a weak (if any) mutagenic activity but are clear inducers of clastogenic damage. ICLs are strongly indicated as key lesions for chromosomal damage whereas oxidized base lesions are unlikely to play a critical role.

Immunoliposome-based targeted delivery of the CRISPR/Cas9gRNA-IL30 complex inhibits prostate cancer and prolongs survival.

The development of selective and nontoxic immunotherapy targeting prostate cancer (PC) is challenging. Interleukin (IL)30 plays immunoinhibitory and oncogenic roles in PC, and its tumor-specific suppression may have significant clinical implications. CRISPR/Cas9-mediated IL30 gene deletion in PC xenografts using anti-PSCA antibody-driven lipid nanocomplexes (Cas9gRNA-hIL30-PSCA NxPs) revealed significant genome editing efficiency and circulation stability without off-target effects or organ toxicity. Biweekly intravenous administration of Cas9gRNA-hIL30-PSCA NxPs to PC-bearing mice inhibited tumor growth and metastasis and improved survival. Mechanistically, Cas9gRNA-hIL30-PSCA NxPs suppressed ANGPTL 1/2/4, IL1ß, CCL2, CXCL1/6, SERPINE1-F1, EFNB2, PLG, PF4, VEGFA, VEGFD, ANG, TGFß1, EGF and HGF expression in human PC cells while upregulated CDH1, DKK3 and PTEN expression, leading to low proliferation and extensive ischemic necrosis. In the syngeneic PC model, IL30-targeting immunoliposomes downregulated NFKB1 expression and prevented intratumoral influx of CD11b+Gr-1+MDCs, Foxp3+Tregs, and NKp46+RORγt+ILC3, and prolonged host survival by inhibiting tumor progression. This study serves as a proof of principle that immunoliposome-based targeted delivery of Cas9gRNA-IL30 represent a potentially safe and effective strategy for PC treatment.

Molecular Targeted Therapies in Metastatic Prostate Cancer: Recent Advances and Future Challenges.

Prostate cancer is the most frequent malignant tumor in men, and, despite the great improvements in survival in patients with localized cancer, the prognosis for metastatic disease remains poor. Novel molecular targeted therapies, which block specific molecules or signaling pathways in tumor cells or in their microenvironment, have shown encouraging results in metastatic castration-resistant prostate cancer. Among these therapeutic approaches, prostate-specific membrane antigen-targeted radionuclide therapies and DNA repair inhibitors represent the most promising ones, with some therapeutic protocols already approved by the FDA, whereas therapies targeting tumor neovascularization and immune checkpoint inhibitors have not yet demonstrated clear clinical benefits. In this review, the most relevant studies and clinical trials on this topic are illustrated and discussed, together with future research directions and challenges.

Interleukin-30 subverts prostate cancer-endothelium crosstalk by fostering angiogenesis and activating immunoregulatory and oncogenic signaling pathways.

BACKGROUND: Cancer-endothelial interplay is crucial for tumor behavior, yet the molecular mechanisms involved are largely unknown. Interleukin(IL)-30, which is expressed as a membrane-anchored cytokine by human prostate cancer (PC) cells, promotes PC vascularization and progression, but the underlying mechanisms have yet to be fully explored. METHODS: PC-endothelial cell (EC) interactions were investigated, after coculture, by flow cytometry, transcriptional profiling, western blot, and ELISA assays. Proteome profiler phospho-kinase array unveiled the molecular pathways involved. The role of tumor-derived IL30 on the endothelium's capacity to generate autocrine circuits and vascular budding was determined following IL30 overexpression, by gene transfection, or its deletion by CRISPR/Cas9 genome editing. Clinical value of the experimental findings was determined through immunopathological study of experimental and patient-derived PC samples, and bioinformatics of gene expression profiles from PC patients. RESULTS: Contact with PC cells favors EC proliferation and production of angiogenic and angiocrine factors, which are boosted by PC expression of IL30, that feeds autocrine loops, mediated by IGF1, EDN1, ANG and CXCL10, and promotes vascular budding and inflammation, via phosphorylation of multiple signaling proteins, such as Src, Yes, STAT3, STAT6, RSK1/2, c-Jun, AKT and, primarily CREB, GSK-3α/ß, HSP60 and p53. Deletion of the IL30 gene in PC cells inhibits endothelial expression of IGF1, EDN1, ANG and CXCL10 and substantially impairs tumor angiogenesis. In its interaction with IL30-overexpressing PC cells the endothelium boosts their expression of a wide range of immunity regulatory genes, including CCL28, CCL4, CCL5, CCR2, CCR7, CXCR4, IL10, IL13, IL17A, FASLG, IDO1, KITLG, TNFA, TNFSF10 and PDCD1, and cancer driver genes, including BCL2, CCND2, EGR3, IL6, VEGFA, KLK3, PTGS1, LGALS4, GNRH1 and SHBG. Immunopathological analyses of PC xenografts and in silico investigation of 1116 PC cases, from the Prostate Cancer Transcriptome Atlas, confirmed the correlation between the expression of IL30 and that of both pro-inflammatory genes, NOS2, TNFA, CXCR5 and IL12B, and cancer driver genes, LGALS4, GNRH1 and SHBG, which was validated in a cohort of 80 PC patients. CONCLUSIONS: IL30 regulates the crosstalk between PC and EC and reshapes their transcriptional profiles, triggering angiogenic, immunoregulatory and oncogenic gene expression programs. These findings highlight the angiostatic and oncostatic efficacy of targeting IL30 to fight PC.

Inactivation of interleukin-30 in colon cancer stem cells via CRISPR/Cas9 genome editing inhibits their oncogenicity and improves host survival.

BACKGROUND: Progression of colorectal cancer (CRC), a leading cause of cancer-related death worldwide, is driven by colorectal cancer stem cells (CR-CSCs), which are regulated by endogenous and microenvironmental signals. Interleukin (IL)-30 has proven to be crucial for CSC viability and tumor progression. Whether it is involved in CRC tumorigenesis and impacts clinical behavior is unknown. METHODS: IL30 production and functions, in stem and non-stem CRC cells, were determined by western blot, immunoelectron microscopy, flow cytometry, cell viability and sphere formation assays. CRISPR/Cas9-mediated deletion of the IL30 gene, RNA-Seq and implantation of IL30 gene transfected or deleted CR-CSCs in NSG mice allowed to investigate IL30's role in CRC oncogenesis. Bioinformatics and immunopathology of CRC samples highlighted the clinical implications. RESULTS: We demonstrated that both CR-CSCs and CRC cells express membrane-anchored IL30 that regulates their self-renewal, via WNT5A and RAB33A, and/or proliferation and migration, primarily by upregulating CXCR4 via STAT3, which are suppressed by IL30 gene deletion, along with WNT and RAS pathways. Deletion of IL30 gene downregulates the expression of proteases, such as MMP2 and MMP13, chemokine receptors, mostly CCR7, CCR3 and CXCR4, and growth and inflammatory mediators, including ANGPT2, CXCL10, EPO, IGF1 and EGF. These factors contribute to IL30-driven CR-CSC and CRC cell expansion, which is abrogated by their selective blockade. IL30 gene deleted CR-CSCs displayed reduced tumorigenicity and gave rise to slow-growing and low metastatic tumors in 80% of mice, which survived much longer than controls. Bioinformatics and CIBERSORTx of the 'Colorectal Adenocarcinoma TCGA Nature 2012' collection, and morphometric assessment of IL30 expression in clinical CRC samples revealed that the lack of IL30 in CRC and infiltrating leucocytes correlates with prolonged overall survival. CONCLUSIONS: IL30 is a new CRC driver, since its inactivation, which disables oncogenic pathways and multiple autocrine loops, inhibits CR-CSC tumorigenicity and metastatic ability. The development of CRISPR/Cas9-mediated targeting of IL30 could improve the current therapeutic landscape of CRC.

CRISPR/Cas9-mediated deletion of Interleukin-30 suppresses IGF1 and CXCL5 and boosts SOCS3 reducing prostate cancer growth and mortality.

BACKGROUND: Metastatic prostate cancer (PC) is a leading cause of cancer death in men worldwide. Targeting of the culprits of disease progression is an unmet need. Interleukin (IL)-30 promotes PC onset and development, but whether it can be a suitable therapeutic target remains to be investigated. Here, we shed light on the relationship between IL30 and canonical PC driver genes and explored the anti-tumor potential of CRISPR/Cas9-mediated deletion of IL30. METHODS: PC cell production of, and response to, IL30 was tested by flow cytometry, immunoelectron microscopy, invasion and migration assays and PCR arrays. Syngeneic and xenograft models were used to investigate the effects of IL30, and its deletion by CRISPR/Cas9 genome editing, on tumor growth. Bioinformatics of transcriptional data and immunopathology of PC samples were used to assess the translational value of the experimental findings. RESULTS: Human membrane-bound IL30 promoted PC cell proliferation, invasion and migration in association with STAT1/STAT3 phosphorylation, similarly to its murine, but secreted, counterpart. Both human and murine IL30 regulated PC driver and immunity genes and shared the upregulation of oncogenes, BCL2 and NFKB1, immunoregulatory mediators, IL1A, TNF, TLR4, PTGS2, PD-L1, STAT3, and chemokine receptors, CCR2, CCR4, CXCR5. In human PC cells, IL30 improved the release of IGF1 and CXCL5, which mediated, via autocrine loops, its potent proliferative effect. Deletion of IL30 dramatically downregulated BCL2, NFKB1, STAT3, IGF1 and CXCL5, whereas tumor suppressors, primarily SOCS3, were upregulated. Syngeneic and xenograft PC models demonstrated IL30's ability to boost cancer proliferation, vascularization and myeloid-derived cell infiltration, which were hindered, along with tumor growth and metastasis, by IL30 deletion, with improved host survival. RNA-Seq data from the PanCancer collection and immunohistochemistry of high-grade locally advanced PCs demonstrated an inverse association (chi-squared test, p = 0.0242) between IL30 and SOCS3 expression and a longer progression-free survival of patients with IL30NegSOCS3PosPC, when compared to patients with IL30PosSOCS3NegPC. CONCLUSIONS: Membrane-anchored IL30 expressed by human PC cells shares a tumor progression programs with its murine homolog and, via juxtacrine signals, steers a complex network of PC driver and immunity genes promoting prostate oncogenesis. The efficacy of CRISPR/Cas9-mediated targeting of IL30 in curbing PC progression paves the way for its clinical use.

Colorectal Cancer-Associated Immune Exhaustion Involves T and B Lymphocytes and Conventional NK Cells and Correlates With a Shorter Overall Survival.

Colorectal cancer (CRC) is one of the most common cancer worldwide, with a growing impact on public health and clinical management. Immunotherapy has shown promise in the treatment of advanced cancers, but needs to be improved for CRC, since only a limited fraction of patients is eligible for treatment, and most of them develop resistance due to progressive immune exhaustion. Here, we identify the transcriptional, molecular, and cellular traits of the immune exhaustion associated with CRC and determine their relationships with the patient's clinic-pathological profile. Bioinformatic analyses of RNA-sequencing data of 594 CRCs from TCGA PanCancer collection, revealed that, in the wide range of immune exhaustion genes, those coding for PD-L1, LAG3 and T-bet were associated (Cramér's V=0.3) with MSI/dMMR tumors and with a shorter overall survival (log-rank test: p=0.0004, p=0.0014 and p=0.0043, respectively), whereas high levels of expression of EOMES, TRAF1, PD-L1, FCRL4, BTLA and SIGLEC6 were associated with a shorter overall survival (log-rank test: p=0.0003, p=0.0188, p=0.0004, p=0.0303, p=0.0052 and p=0.0033, respectively), independently from the molecular subtype of CRC. Expression levels of PD-L1, PD-1, LAG3, EOMES, T-bet, and TIGIT were significantly correlated with each other and associated with genes coding for CD4+ and CD8+CD3+ T cell markers and NKp46+CD94+EOMES+T-bet+ cell markers, (OR >1.5, p<0.05), which identify a subset of group 1 innate lymphoid cells, namely conventional (c)NK cells. Expression of TRAF1 and BTLA co-occurred with both T cell markers, CD3γ, CD3δ, CD3ε, CD4, and B cell markers, CD19, CD20 and CD79a (OR >2, p<0.05). Expression of TGFß1 was associated only with CD4+ and CD8+CD3ε+ T cell markers (odds ratio >2, p<0.05). Expression of PD-L2 and IDO1 was associated (OR >1.5, p<0.05) only with cNK cell markers, whereas expression of FCRL4, SIGLEC2 and SIGLEC6 was associated (OR >2.5; p<0.05) with CD19+CD20+CD79a+ B cell markers. Morphometric examination of immunostained CRC tissue sections, obtained from a validation cohort of 53 CRC patients, substantiated the biostatistical findings, showing that the highest percentage of immune exhaustion gene expressing cells were found in tumors from short-term survivors and that functional exhaustion is not confined to T lymphocytes, but also involves B cells, and cNK cells. This concept was strengthened by CYBERSORTx analysis, which revealed the expression of additional immune exhaustion genes, in particular FOXP1, SIRT1, BATF, NR4A1 and TOX, by subpopulations of T, B and NK cells. This study provides novel insight into the immune exhaustion landscape of CRC and emphasizes the need for a customized multi-targeted therapeutic approach to overcome resistance to current immunotherapy.

Interleukin-30 feeds breast cancer stem cells via CXCL10 and IL23 autocrine loops and shapes immune contexture and host outcome.

BACKGROUND: Breast cancer (BC) progression to metastatic disease is the leading cause of death in women worldwide. Metastasis is driven by cancer stem cells (CSCs) and signals from their microenvironment. Interleukin (IL) 30 promotes BC progression, and its expression correlates with disease recurrence and mortality. Whether it acts by regulating BCSCs is unknown and could have significant therapeutic implications. METHODS: Human (h) and murine (m) BCSCs were tested for their production of and response to IL30 by using flow cytometry, confocal microscopy, proliferation and sphere-formation assays, and PCR array. Immunocompetent mice were used to investigate the role of BCSC-derived IL30 on tumor development and host outcome. TCGA PanCancer and Oncomine databases provided gene expression data from 1084 and 75 hBC samples, respectively, and immunostaining unveiled the BCSC microenvironment. RESULTS: hBCSCs constitutively expressed IL30 as a membrane-anchored glycoprotein. Blocking IL30 hindered their proliferation and self-renewal efficiency, which were boosted by IL30 overexpression. IL30 regulation of immunity gene expression in human and murine BCSCs shared a significant induction of IL23 and CXCL10. Both immunoregulatory mediators stimulated BCSC proliferation and self-renewal, while their selective blockade dramatically hindered IL30-dependent BCSC proliferation and mammosphere formation. Orthotopic implantation of IL30-overexpressing mBCSCs, in syngeneic mice, gave rise to poorly differentiated and highly proliferating MYC+KLF4+LAG3+ tumors, which expressed CXCL10 and IL23, and were infiltrated by myeloid-derived cells, Foxp3+ T regulatory cells and NKp46+RORγt+ type 3 innate lymphoid cells, resulting in increased metastasis and reduced survival. In tumor tissues from patients with BC, expression of IL30 overlapped with that of CXCL10 and IL23, and ranked beyond the 95th percentile in a Triple-Negative enriched BC collection from the Oncomine Platform. CIBERSORTx highlighted a defective dendritic cell, CD4+ T and γδ T lymphocyte content and a prominent LAG3 expression in IL30highversus IL30low human BC samples from the TCGA PanCancer collection. CONCLUSIONS: Constitutive expression of membrane-bound IL30 regulates BCSC viability by juxtacrine signals and via second-level mediators, mainly CXCL10 and IL23. Their autocrine loops mediate much of the CSC growth factor activity of IL30, while their paracrine effect contributes to IL30 shaping of immune contexture. IL30-related immune subversion, which also emerged from computational analyses, strongly suggests that targeting IL30 can restrain the BCSC compartment and counteract BC progression.

The C-X-C Motif Chemokine Ligand 1 Sustains Breast Cancer Stem Cell Self-Renewal and Promotes Tumor Progression and Immune Escape Programs.

Breast cancer (BC) mortality is mainly due to metastatic disease, which is primarily driven by cancer stem cells (CSC). The chemokine C-X-C motif ligand-1 (CXCL1) is involved in BC metastasis, but the question of whether it regulates breast cancer stem cell (BCSC) behavior is yet to be explored. Here, we demonstrate that BCSCs express CXCR2 and produce CXCL1, which stimulates their proliferation and self-renewal, and that CXCL1 blockade inhibits both BCSC proliferation and mammosphere formation efficiency. CXCL1 amplifies its own production and remarkably induces both tumor-promoting and immunosuppressive factors, including SPP1/OPN, ACKR3/CXCR7, TLR4, TNFSF10/TRAIL and CCL18 and, to a lesser extent, immunostimulatory cytokines, including IL15, while it downregulates CCL2, CCL28, and CXCR4. CXCL1 downregulates TWIST2 and SNAI2, while it boosts TWIST1 expression in association with the loss of E-Cadherin, ultimately promoting BCSC epithelial-mesenchymal transition. Bioinformatic analyses of transcriptional data obtained from BC samples of 1,084 patients, reveals that CXCL1 expressing BCs mostly belong to the Triple-Negative (TN) subtype, and that BC expression of CXCL1 strongly correlates with that of pro-angiogenic and cancer promoting genes, such as CXCL2-3-5-6, FGFBP1, BCL11A, PI3, B3GNT5, BBOX1, and PTX3, suggesting that the CXCL1 signaling cascade is part of a broader tumor-promoting signaling network. Our findings reveal that CXCL1 functions as an autocrine growth factor for BCSCs and elicits primarily tumor progression and immune escape programs. Targeting the CXCL1/CXCR2 axis could restrain the BCSC compartment and improve the treatment of aggressive BC.

Hindering triple negative breast cancer progression by targeting endogenous interleukin-30 requires IFNγ signaling.

IL30mRNA expression is associated with the TNBC subtype. IL30 boosts proliferation and migration of TNBC cells and reshapes their immunity gene expression profile. The lack of endogenous IL30 hinders TNBC growth and progression and prolongs host survival. TNBC growth inhibition, due to the lack of endogenous IL30, requires INFγ production by T and NK cells.

NOTCH1 Activation Negatively Impacts on Chronic Lymphocytic Leukemia Outcome and Is Not Correlated to the NOTCH1 and IGHV Mutational Status.

NOTCH1 mutations and deregulated signal have been commonly found in chronic lymphocytic leukemia (CLL) patients. Whereas the impact of NOTCH1 mutations on clinical course of CLL has been widely studied, the prognostic role of NOTCH1 activation in CLL remains to be defined. Here, we analyzed the activation of NOTCH1/NOTCH2 (ICN1/ICN2) and the expression of JAGGED1 (JAG1) in 163 CLL patients and evaluated their impact on TTFT (Time To First Treatment) and OS (Overall Survival). NOTCH1 activation (ICN1+) was found in 120/163 (73.6%) patients. Among them, 63 (52.5%) were NOTCH1 mutated (ICN1+/mutated) and 57 (47.5%) were NOTCH1 wild type (ICN1+/WT). ICN1+ patients had a significant reduction of TTFT compared to ICN1-negative (ICN1-). In the absence of NOTCH1 mutations, we found that the ICN1+/WT group had a significantly reduced TTFT compared to ICN1- patients. The analysis of IGHV mutational status showed that the distribution of the mutated/unmutated IGHV pattern was similar in ICN1+/WT and ICN1- patients. Additionally, TTFT was significantly reduced in ICN1+/ICN2+ and ICN1+/JAG1+ patients compared to ICN1-/ICN2- and ICN1-/JAG1- groups. Our data revealed for the first time that NOTCH1 activation is a negative prognosticator in CLL and is not correlated to NOTCH1 and IGHV mutational status. Activation of NOTCH2 and JAGGED1 expression might also influence clinical outcomes in this group, indicating the need for further dedicated studies. The evaluation of different NOTCH network components might represent a new approach to refine CLL risk stratification.

The lack of epithelial interleukin-7 and BAFF/BLyS gene expression in prostate cancer as a possible mechanism of tumor escape from immunosurveillance.

PURPOSE: The human prostate is endowed with intraepithelial and stromal lymphocytes, which may develop lymphoid follicles (LF) and allow a local immune response. We sought to investigate whether interleukin (IL)-7 and BAFF/BLyS, two fundamental survival factors for T and B cells, are expressed in the normal and neoplastic prostate and affect intraprostatic lymphocyte homeostasis. EXPERIMENTAL DESIGN: We have used real-time reverse transcription-PCR of microdissected prostatic glands and confocal microscopy to detect cytokine production, combined with immunohistochemistry to characterize intraprostatic lymphocytes. RESULTS: Prostatic epithelia constitutively produce IL-7 and, to a lesser extent, BAFF/BLyS. Indeed, we show that IL-7 receptor alpha is expressed by intraepithelial T lymphocytes and parafollicular T cells, whereas BAFF-R is found on periglandular B lymphocytes and mantle zone B cells of LFs. Prostate-homing B and T lymphocytes are scarcely proliferating, whereas most of them express the antiapoptotic protein bcl-2 and reveal a low apoptotic index in the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The transition from normal to neoplastic glands in prostate cancer (PCa) is marked by a dramatic decline of IL-7 and BAFF/BLyS production. Accordingly, PCa is characterized by a significant reduction of intraepithelial lymphocytes and loss of LFs. B-cell and T-cell expression of bcl-2 decrease, whereas the apoptotic events increase. The remaining PCa-infiltrating lymphocytes are mostly CD8(+) T cells that lack terminal differentiation and barely penetrate neoplastic glands. CONCLUSIONS: These results suggest that epithelial IL-7 and BAFF/BLyS production support intraprostatic lymphocyte survival. Its loss in PCa is associated with a severe depletion of prostate-associated lymphocytes and points to a novel tumor escape mechanism.

Expression of IL-32 in human lung cancer is related to the histotype and metastatic phenotype.

RATIONALE: A strong link has been recently demonstrated between inflammation and lung cancer. Thus, we investigated whether the proinflammatory cytokine IL-32 may be involved in lung carcinogenesis and hence provide a novel therapeutic target. OBJECTIVES: Lung cancer subtypes display different clinical outcomes. We have set out to clarify the role of IL-32 in the physiopathology of the main histotypes. METHODS: IL-32 expression, as visualized by immunohistochemistry on 23 premalignant and 148 malignant lesions, was correlated with clinicopathological and survival data. Confocal microscopy, microdissection, and real-time reverse transcription-polymerase chain reaction were used to identify cell sources and expression levels of IL-32. MEASUREMENTS AND MAIN RESULTS: IL-32 expression was lacking in the majority of squamous-cell carcinomas (SCC) (76%) and their precursor lesions, but strongly up-regulated in most adenocarcinomas (AC) (73%) and their precursors, 64% of large-cell carcinomas, and 77% of small-cell lung cancers. Lymph node metastases frequently developed from IL-32-expressing lung cancers, and especially (82%) from those endowed with an IL-32-expressing leukocyte infiltrate (TIL) mainly composed of CD68(+) macrophages, CD4(+) T lymphocytes, and DC-SIGN(+) dendritic cells. Expression levels of IL-32 by both TIL and tumor cells (TC), particularly in AC and SCC, were paralleled by those of IL-6, IL-8, and vascular endothelial growth factor in the same cell population and correlated with high intratumor microvessel density and poor clinical outcome. CONCLUSIONS: IL-32 is probably implicated in the pathogenesis of most lung cancer histotypes but unlikely in that of SCC. Its TIL and TC expression are both associated with acquisition of an invasive and metastatic phenotype and may be a useful prognostic indicator.

Erratum to clinical impact of the lung tissue transcriptome in a teenager with multifocal invasive mucinous adenocarcinoma-a case report.

[This corrects the article DOI: 10.21037/tlcr-20-177.].

Clinical impact of the lung tissue transcriptome in a teenager with multifocal invasive mucinous adenocarcinoma-a case report.

The transcriptional profiling of cancer and normal tissues harboring cancer can be a clinical and discovery tool, especially for the study of rare tumors. Invasive mucinous adenocarcinoma (IMA) is a rare lung cancer histotype, which mostly affects the elderly and commonly has a poor prognosis. We investigated the exceptional case of a teenager, exposed to passive smoke and chemical carcinogens, who developed a multifocal IMA with bilateral involvement. The malignancy was asymptomatic and was diagnosed occasionally during hospitalization for acute abdominal pain due to adnexitis. The young patient underwent video-assisted thoracoscopic surgery and lung samples were analysed by RNA-Sequencing. The transcriptome of patient's normal and neoplastic lung tissues was compared with matched healthy controls and IMA signature cases, using Gene Set Enrichment Analyses, Gene Ontology and Genotype Tissue Expression database. Compared to healthy controls, the patient's lung tissue lacked the expression of lymphocyte and humoral-mediated immune response genes, whereas genes driving the response to stimulus, chemical and organic substances, primarily, CXCL8, ACKR1, RAB7B, HOXC9, HOXD9, KLF5 and NKX2-8 were overexpressed. Genes driving extracellular structure organization, cell adhesion, cell movement, metabolic and apoptotic processes were down-modulated in patient's lung tissue. When compared to IMA signature cases, the patient's IMA revealed a prevalent expression of genes regulating the response to stimulus, myeloid and neutrophil activation and immune system processes, primarily CD1a and CXCL13/BCA1, whereas stemness genes and proto-oncogenes, such as SOX4, HES1, IER3 and SERPINH1 were downmodulated. These transcriptional signature associated with a favorable clinical course, since the patient was healthy five years after initial diagnosis. The transcriptome of the normal tissues bearing tumor provides meaningful information on the gene pathways driving tumor histogenesis, with a prospective impact on early diagnosis. Unlike the tumor histotype-related transcriptional signature, the individual patient's signature enables tailored treatment and accurate prognosis.

Intraocular Biopsy and ImmunoMolecular Pathology for "Unmasking" Intraocular Inflammatory Diseases.

Intraocular inflammation can hide a variety of eye pathologies. In 33% of cases, to obtain a correct diagnosis, investigation of the intraocular sample is necessary. The combined analyses of the intraocular biopsy, using immuno-pathology and molecular biology, point to resolve the diagnostic dilemmas in those cases where history, clinical tests, and ophthalmic and systemic examinations are inconclusive. In such situations, the teamwork between the ophthalmologist and the molecular pathologist is critically important to discriminate between autoimmune diseases, infections, and intraocular tumors, including lymphoma and metastases, especially in those clinical settings known as masquerade syndromes. This comprehensive review focuses on the diagnostic use of intraocular biopsy and highlights its potential to enhance research in the field. It describes the different surgical techniques of obtaining the biopsy, risks, and complication rates. The review is organized according to the anatomical site of the sample: I. anterior chamber containing aqueous humor, II. iris and ciliary body, III. vitreous, and IV. choroid and retina. We have excluded the literature concerning biopsy for choroidal melanoma and retinoblastoma, as this is a specialized area more relevant to ocular oncology.

Targeting Interleukin(IL)-30/IL-27p28 signaling in cancer stem-like cells and host environment synergistically inhibits prostate cancer growth and improves survival.

BACKGROUND: Interleukin(IL)-30/IL-27p28 production by Prostate Cancer (PC) Stem-Like Cells (SLCs) has proven, in murine models, to be critical to tumor onset and progression. In PC patients, IL-30 expression by leukocytes infiltrating PC and draining lymph nodes correlates with advanced disease grade and stage. Here, we set out to dissect the role of host immune cell-derived IL-30 in PC growth and patient outcome. METHODS: PC-SLCs were implanted in wild type (WT) and IL-30 conditional knockout (IL-30KO) mice. Histopathological and cytofluorimetric analyses of murine tumors and lymphoid tissues prompted analyses of patients' PC samples and follow-ups. RESULTS: Implantation of PC-SLCs in IL-30KO mice, gave rise to slow growing tumors characterized by apoptotic events associated with CD4+T lymphocyte infiltrates and lack of CD4+Foxp3+ T regulatory cells (Tregs). IL-30 knockdown in PC-SLCs reduced cancer cell proliferation, vascularization and intra-tumoral Indoleamine 2,3-Dioxygenase (IDO)+CD11b+Gr-1+ myeloid-derived cells (MDCs) and led to a significant delay in tumor growth and increase in survival. IL-30-silenced tumors developed in IL-30KO mice, IL-30-/-tumors, lacked vascular supply and displayed frequent apoptotic cancer cells entrapped by perforin+TRAIL+CD3+Tlymphocytes, most of which had a CD4+T phenotype, whereas IL-10+TGFß+Foxp3+Tregs were lacking. IL-30 silencing in PC-SLCs prevented lung metastasis in 73% of tumor-bearing WT mice and up to 80% in tumor-bearing IL-30KO mice. In patients with high-grade and locally advanced PC, those with IL-30-/-tumors, showed distinct intra-tumoral cytotoxic granule-associated RNA binding protein (TIA-1)+CD4+Tlymphocyte infiltrate, rare Foxp3+Tregs and a lower biochemical recurrence rate compared to patients with IL-30+/+tumors in which IL-30 is expressed in both tumor cells and infiltrating leukocytes. CONCLUSION: The lack of host leukocyte-derived IL-30 inhibits Tregs expansion, promotes intra-tumoral infiltration of CD4+T lymphocytes and cancer cell apoptosis. Concomitant lack of MDC influx, obtained by IL-30 silencing in PC-SLCs, boosts cytotoxic T lymphocyte activation and cancer cell apoptosis resulting in a synergistic tumor suppression with the prospective benefit of better survival for patients with advanced disease.

Crucial pathophysiological role of CXCR2 in experimental ulcerative colitis in mice.

Polymorphonuclear leukocyte infiltration and activation into colonic mucosa are believed to play a pivotal role in mediating tissue damage in human ulcerative colitis (UC). Ligands of human CXC chemokine receptor 1 and 2 (CXCR1/R2) are chemoattractants of PMN, and high levels were found in the mucosa of UC patients. To investigate the pathophysiological role played by CXCR2 in experimental UC, we induced chronic experimental colitis in WT and CXCR2(-/-) mice by two consecutive cycles of 4% dextran sulfate sodium administration in drinking water. In wild-type (WT) mice, the chronic relapsing of DSS-induced colitis was characterized by clinical signs and histopathological findings that closely resemble human disease. CXCR2(-/-) mice failed to show PMN infiltration into the mucosa and, consistently with a key role of PMN in mediating tissue damage in UC, showed limited signs of mucosal damage and reduced clinical symptoms. Our data demonstrate that CXCR2 plays a key pathophysiological role in experimental UC, suggesting that CXCR2 activation may represent a relevant pharmacological target for the design of novel pharmacological treatments in human UC.