An HGF-dependent positive feedback loop between bladder cancer cells and fibroblasts mediates lymphangiogenesis and lymphatic metastasis

Cancer-associated fibroblasts (CAFs) are essential etiologic actors in promoting tumor progression via extensive reciprocal interactions with cancer cells. Yet, the biological role and regulatory mechanism of CAFs phenotype underlying lymph node (LN) metastasis of bladder cancer (BCa) remain unclear. Here, we report that BCa cell-secreted extracellular vesicles (EVs) played an important role in the CAF-enriched microenvironment, which correlated with BCa lymphangiogenesis and LN metastasis. RNA sequencing identified an EV-associated long noncoding RNA, LINC00665, which acted as a crucial mediator of CAF infiltration in BCa. LINC00665 mediated EV release from BCa cells to endow fibroblasts with the CAF phenotype, which reciprocally induced LINC00665 upregulation to form a RAB27B-HGF-c-Myc positive feedback loop, facilitating BCa lymphangiogenesis and LN metastasis. Importantly, we demonstrate that Cabozantinib significantly suppressed LINC00665-mediated BCa LN metastasis in an orthotopic xenograft model. Our study highlights a molecular mechanism by which LINC00665 induces a RAB27B-HGF-c-Myc positive feedback loop between cancer cells and fibroblasts to sustain BCa LN metastasis, and represents LINC00665 as a potential therapeutic target in BCa LN metastasis.

STEM-15. SerpinB3 DRIVES CANCER STEM CELL SURVIVAL IN GLIOBLASTOMA

Despite therapeutic interventions for glioblastoma (GBM), self-renewing, therapy-resistant populations of cells referred to as cancer stem cells (CSCs) drive recurrence. Previously, we identified the unique expression of junctional adhesion molecule-A (JAM-A) on CSCs and demonstrated that JAM-A is both necessary and sufficient for self-renewal and tumor growth. Moreover, we determined that JAM-A signals via Akt in GBM CSCs to sustain pluripotency transcription factor activity; however, the entire signaling network has yet to be fully elucidated. To further delineate this pathway, we immunoprecipitated JAM-A from patient-derived GBM CSCs and performed mass spectrometry to determine JAM-A binding proteins. This led to the identification of the cysteine protease inhibitor SerpinB3 as a putative JAM-A binding partner. Using in vitro CSC functional assays, we show that SerpinB3 is necessary for CSC maintenance and survival. In an in vivo orthotopic xenograft model, knockdown of SerpinB3 extended survival. Mechanistically, knockdown of SerpinB3 led to decreased expression of TGF-β, Myc, WNT, and Notch signaling, known regulators of the CSC state. Additionally, knockdown of SerpinB3 increases susceptibility to radiation therapy. SerpinB3 is essential for buffering cells against cathepsin-mediated cell death, and we found that elevated lysosomal membrane permeability after radiation leads to cathepsin release into the cytoplasm. As a result, SerpinB3 knockdown cells have a diminished capacity to inhibit cathepsin-driven cell death after radiation. The addition of the cathepsin inhibitor E64D partially rescues the SerpinB3 knockdown, however, SerpinB3 mutants that are unable to inhibit cathepsins fail to do the same. Taken together, our findings, identify a novel GBM CSC-specific survival mechanism involving a previously uninvestigated cysteine protease inhibitor, SerpinB3, and provide a potential target to increase the efficacy of standard of care GBM therapies against therapy-resistant CSCs.

LRP-1 Matricellular Receptor Involvement in Triple Negative Breast Cancer Tumor Angiogenesis

Background: LRP-1 is a multifunctional scavenger receptor belonging to the LDLR family. Due to its capacity to control pericellular levels of various growth factors and proteases, LRP-1 plays a crucial role in membrane proteome dynamics, which appears decisive for tumor progression. Methods: LRP-1 involvement in a TNBC model was assessed using an RNA interference strategy in MDA-MB-231 cells. In vivo, tumorigenic and angiogenic effects of LRP-1-repressed cells were evaluated using an orthotopic xenograft model and two angiogenic assays (Matrigel® plugs, CAM). DCE-MRI, FMT, and IHC were used to complete a tumor longitudinal follow-up and obtain morphological and functional vascular information. In vitro, HUVECs’ angiogenic potential was evaluated using a tumor secretome, subjected to a proteomic analysis to highlight LRP-1-dependant signaling pathways. Results: LRP-1 repression in MDA-MB-231 tumors led to a 60% growth delay because of, inter alia, morphological and functional vascular differences, confirmed by angiogenic models. In vitro, the LRP-1-repressed cells secretome restrained HUVECs’ angiogenic capabilities. A proteomics analysis revealed that LRP-1 supports tumor growth and angiogenesis by regulating TGF-β signaling and plasminogen/plasmin system. Conclusions: LRP-1, by its wide spectrum of interactions, emerges as an important matricellular player in the control of cancer-signaling events such as angiogenesis, by supporting tumor vascular morphology and functionality.

BKM120 sensitizes glioblastoma to the PARP inhibitor rucaparib by suppressing homologous recombination repair

PARP inhibitors have been approved for the therapy of cancers with homologous recombination (HR) deficiency based on the concept of “synthetic lethality”. However, glioblastoma (GBM) patients have gained little benefit from PARP inhibitors due to a lack of BRCA mutations. Herein, we demonstrated that concurrent treatment with the PARP inhibitor rucaparib and the PI3K inhibitor BKM120 showed synergetic anticancer effects on GBM U251 and U87MG cells. Mechanistically, BKM120 decreased expression of HR molecules, including RAD51 and BRCA1/2, and reduced HR repair efficiency in GBM cells, therefore increasing levels of apoptosis induced by rucaparib. Furthermore, we discovered that the two compounds complemented each other in DNA damage response and drug accumulation. Notably, in the zebrafish U87MG-RFP orthotopic xenograft model, nude mouse U87MG subcutaneous xenograft model and U87MG-Luc orthotopic xenograft model, combination showed obviously increased antitumor efficacy compared to each monotherapy. Immunohistochemical analysis of tumor tissues indicated that the combination obviously reduced expression of HR repair molecules and increased the DNA damage biomarker γ-H2AX, consistent with the in vitro results. Collectively, our findings provide new insight into combined blockade of PI3K and PARP, which might represent a promising therapeutic approach for GBM.

miRNA-34c suppresses osteosarcoma progression in vivo by targeting Notch and E2F

The expression of microRNAs (miRNAs) is dysregulated in many types of cancers including osteosarcoma (OS) due to genetic and epigenetic alterations. Among these, miR-34c, an effector of tumor suppressor P53 and an upstream negative regulator of Notch signaling in osteoblast differentiation, is dysregulated in OS. Here, we demonstrated a tumor suppressive role of miR-34c in OS progression using in vitro assays and in vivo genetic mouse models. We found that miR-34c inhibits the proliferation and the invasion of metastatic OS cells, which resulted in reduction of the tumor burden and increased overall survival in an orthotopic xenograft model. Moreover, the osteoblast specific over expression of miR-34c increased survival in the osteoblast specific p53 mutant OS mouse model. We found that miR-34c regulates the transcription of several genes in Notch signaling (NOTCH1, JAG1 and HEY2) and in p53 mediated cell cycle and apoptosis (CCNE2, E2F5, E2F2 and HDAC1). More interestingly, we found that the metastatic free survival probability was increased among a patient cohort from TARGET OS which has lower expression of direct targets of miR-34c that was identified in our transcriptome analysis such as E2F5 and NOTCH1. In conclusion, we demonstrate that miR-34c is a tumor suppressive miRNA in OS progression in vivo. In addition, we highlight the therapeutic potential of targeting miR-34c in OS.

Trabectedin (T) in desmoplastic small round cell tumor (DSRCT): Report of its effect in 3 relapsed patients (pts) and the comparison of different regimens in a patient-derived xenograft (PDX) model.

e23553 Background: DSRCT is an ultra-rare soft tissue sarcoma marked by the presence of the EWS-WT1 translocation and a dismal prognosis. Anecdotal activity of T in DSRCT pts was reported. We describe herein three advanced DSRCT pts treated with T and a comparative assessment of doxorubicin (D), pazopanib (P) and T in a patient-derived xenograft (PDX) model of DSRCT. Methods: Three pts (#1, #2 and #3) suffering from progressive, metastatic, unresectable relapsing disease from a primary peritoneal DSRCT previously treated with 8 cycles of anthracycline-based neoadjuvant chemo and complete surgical resection, were started on T (1.3 mcg/sqm every 3-4 weeks). A PDX model was generated by subcutaneously implanting small tumor fragments obtained at surgery from a treatment-naïve DSRCT patient into the right flank of SCID mice. Consistency of PDX and the originating tumor was confirmed in terms of histomorphology and presence of the EWS-WT1 gene fusion. Mice were randomized to receive D, P and T, administered as single agents at optimal doses and schedules. Drug activity was assessed in terms of tumor volume inhibition (TVI) percentage in treated versus control mice. An orthotopic xenograft model was also generated by injecting DSRCT cells into the peritoneal cavity of SCID mice. Results: At the time of this report, pt #1 and #2 are on therapy with T, with a partial response by RECIST maintained after 48 and 36 months from treatment start, respectively, while #3 progressed after 4 months. In the DSRCT PDX model, T was the most effective drug, with a maximum TVI of 82%, while D and P showed lower, comparable activity (maximum TVI: 59% and 66%, respectively). In the orthotopic DSRCT PDX, DSRCT cells spreading in the abdominal cavity generated different tumor masses, properly recapitulating the dissemination pattern in patients, confirming the reliability of this preclinical model. Conclusions: Both our preliminary model and our further clinical observations support the potential of T in DSRCT. A confirmatory prospective clinical study is now warranted.

Development of human hepatocellular carcinoma in X-linked severe combined immunodeficient pigs: An orthotopic xenograft model

Hepatocellular carcinoma (HCC) is the fifth most common primary tumor and the third leading cause of cancer-related deaths worldwide. Rodent models of HCC have contributed to the advancement of studies investigating liver carcinogenesis, tumor-host interactions, and drug screening. However, their small size renders them unsuitable for surgical or clinical imaging studies, necessitating the development of larger-size HCC models. Here, we developed a xenograft model of human HCC in X-linked interleukin-2 receptor gamma chain gene (Il2rg)-targeted severe combined immunodeficient (SCID) pigs. HepG2 cell suspension in serum-free medium containing 50% membrane matrix was directly injected into the liver parenchyma of eight X-linked Il2rg-targeted SCID pigs (6.6–15.6 kg) via ultrasonography-guided percutaneous puncture. Tumor engraftment was evaluated weekly using ultrasonography, and cone-beam computed tomography was performed during arterial portography (CTAP) and hepatic arteriography (CTHA) to evaluate the hemodynamics of engrafted tumors. The engrafted tumors were histologically analyzed following necropsy and assessed for pathological similarities to human HCCs. Macroscopic tumor formation was observed in seven of the eight pigs (simple nodular tumors in three and multinodular tumors in four). Engrafted tumors were identified as low-echoic upon ultrasonography and as perfusion-defect nodules on the CTAP images. Meanwhile, CTHA showed that the tumors were hyperattenuating. Further, histopathological findings of the engrafted tumors were consistent with those of human HCC. In conclusion, the porcine model of human HCC, successfully generated herein, might help develop more effective therapeutic strategies for HCC.

Glypican-3 targeted delivery of 89Zr and 90Y as a theranostic radionuclide platform for hepatocellular carcinoma

Glypican-3 (GPC3) is a tumor associated antigen expressed by hepatocellular carcinoma (HCC) cells. This preclinical study evaluated the efficacy of a theranostic platform using a GPC3-targeting antibody αGPC3 conjugated to zirconium-89 (89Zr) and yttrium-90 (90Y) to identify, treat, and assess treatment response in a murine model of HCC. A murine orthotopic xenograft model of HCC was generated. Animals were injected with 89Zr-labeled αGPC3 and imaged with a small-animal positron emission/computerized tomography (PET/CT) imaging system (immuno-PET) before and 30 days after radioimmunotherapy (RIT) with 90Y-labeled αGPC3. Serum alpha fetoprotein (AFP), a marker of tumor burden, was measured. Gross tumor volume (GTV) and SUVmax by immuno-PET was measured using fixed intensity threshold and manual segmentation methods. Immuno-PET GTV measurements reliably quantified tumor burden prior to RIT, strongly correlating with serum AFP (R2 = 0.90). Serum AFP was significantly lower 30 days after RIT in 90Y-αGPC3 treated animals compared to those untreated (p = 0.01) or treated with non-radiolabeled αGPC3 (p = 0.02). Immuno-PET GTV measurements strongly correlated with tumor burden after RIT (R2 = 0.87), and GTV of animals treated with 90Y-αGPC3 was lower than in animals who did not receive treatment or were treated with non-radiolabeled αGPC3, although this only trended toward statistical significance. A theranostic platform utilizing GPC3 targeted 89Zr and 90Y effectively imaged, treated, and assessed response after radioimmunotherapy in a GPC3-expressing HCC xenograft model.

Selective exosome exclusion of miR-375 by glioma cells promotes glioma progression by activating the CTGF-EGFR pathway

Background Exosomes are membrane-bound extracellular vesicles of 40–150 nm in size, that are produced by many cell types, and play an important role in the maintenance of cellular homeostasis. Exosome secretion allows for the selective removal of harmful substances from cells. However, it remains unclear whether this process also takes place in glioma cells. Methods Herein, the role of the tumour-suppressor miR-375 was explored in human glioma cells. Immunoblotting and qRT-PCR experiments demonstrated a functional link between miR-375 and its target, connectivetissuegrowthfactor (CTGF), which led to the identification of the underlying molecular pathways. The exosomes secreted by glioma cells were extracted by ultracentrifugation and examined by transmission electron microscopy. Exosomal expression of miR-375 was then analysed by qRT-PCR; while the exosome secretion inhibitor, GW4869, was used to examine the biological significance of miR-375 release. Moreover, the dynamics of miR-375 release by glioma cells was investigated using fluorescently labelled exosomes. Finally, exosomal miR-375 release was examined in an orthotopic xenograft model in nude mice. Results MiR-375 expression was downregulated in gliomas. MiR-375 suppressed glioma proliferation, migration, and invasion by inhibiting the CTGF-epidermalgrowthfactorreceptor (EGFR) signalling pathway. MiR-375-containing exosomes were also identified in human peripheral blood samples from glioma patients, and their level correlated with disease progression status. Exosomal miR-375 secretion impacted the CTGF-EGFR pathway activity. Once secreted, exosomal miR-375 was not taken back up by glioma cells. Conclusions Exosomal miR-375 secretion allowed for sustained activation of the CTGF-EGFR oncogenic pathway, promoting the proliferation and invasion of glioma cells. These findings enhance our understanding of exosome biology and may inspire development of new glioma therapies.