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-30 (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 the human IL30 (hIL30) gene and functionalized with anti-PSCA-Abs (Cas9hIL30-PSCA NPs). Efficiency of the NPs in targeting IL-30 and the metastatic potential of PC cells was examined in vivo in xenograft models of lung metastasis, and in vitro by using two organ-on-chip (2-OC)-containing 3D spheroids of IL30+ PC-endothelial cell 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 tumor microemboli substantially hindered lung metastasization. Cas9hIL30-PSCA NPs inhibited PC cell proliferation and expression of IL-30 and metastasis drivers, such as CXCR2, CXCR4, IGF1, L1CAM, METAP2, MMP2, and TNFSF10, whereas CDH1 was upregulated. PC-Lung and PC-BM 2-OCs revealed that Cas9hIL30-PSCA NPs suppressed PC cell release of CXCL2/GROβ, which was associated with intra-metastatic myeloid cell infiltrates, and of DKK1, OPG, and IL-6, which boosted endothelial network formation and cancer cell migration. Development of a patient-tailored nanoplatform for selective CRISPR-mediated IL-30 gene deletion is a clinically valuable tool against PC progression.

Fieni C ，Ciummo SL ，Sorrentino C ，Marchetti S ，Vespa S ，Lanuti P ，Lotti LV ，Di Carlo E ... -  《-》

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, IL1β, CCL2, CXCL1/6, SERPINE1-F1, EFNB2, PLG, PF4, VEGFA, VEGFD, ANG, TGFβ1, 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.

Fieni C ，Sorrentino C ，Ciummo SL ，Fontana A ，Lotti LV ，Scialis S ，Calvo Garcia D ，Caulo M ，Di Carlo E ... -  《-》

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

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. 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. 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. 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.

Sorrentino C ，D'Antonio L ，Ciummo SL ，Fieni C ，Landuzzi L ，Ruzzi F ，Vespa S ，Lanuti P ，Lotti LV ，Lollini PL ，Di Carlo E ... -  《Journal of Hematology & Oncology》

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

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. 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. 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/β, 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. 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.

Ciummo SL ，Sorrentino C ，Fieni C ，Di Carlo E ... -  《JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH》

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

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. 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. 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. 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.

D'Antonio L ，Fieni C ，Ciummo SL ，Vespa S ，Lotti L ，Sorrentino C ，Di Carlo E ... -  《Journal for ImmunoTherapy of Cancer》