Low-protein diet during pregnancy enhances ZIKV’s ability to produce congenital abnormalities.

The Zika virus (ZIKV) has shown a promising oncolytic effect against embryonal CNS tumors. However, studies on the effect of different administration routes and the ideal viral load in preclinical models are highly relevant aiming for treatment safety and efficiency. Here, we investigated the effect and effectiveness of different routes of administration, and the number of ZIKV^BR injections on tumor tropism, destruction, and side effects. Furthermore, we designed an early-stage human brain organoid co-cultured with embryonal CNS tumors to analyze the ZIKV^BR oncolytic effect. We showed that in the mice bearing subcutaneous tumors, the ZIKV^BR systemically presented a tropism to the brain. When the tumor was located in the mice’s brain, serial systemic injections presented efficient tumor destruction, with no neurological or other organ injury and increased mice survival. In the human cerebral organoid model co-cultured with embryonal CNS tumor cells, ZIKV^BR impaired tumor progression. The gene expression of cytokines and chemokines in both models suggested an enhancement of immune cells recruitment and tumor inflammation after the treatment. These results open new perspectives for virotherapy using the ZIKV^BR systemic administration route and multiple doses of low virus load for safe and effective treatment of embryonal CNS tumors, an orphan disease that urges new effective therapies.

Central nervous system (CNS) cancers are responsible for high rates of morbidity and mortality worldwide. Malignant CNS tumors such as adult Glioblastoma (GBM) and pediatric embryonal CNS tumors such as medulloblastoma (MED) and atypical teratoid rhabdoid tumors (ATRT) present relevant therapeutic challenges due to the lack of response to classic treatment regimens with radio and chemotherapy. Recent findings on the Zika virus’ (ZIKV) ability to infect and kill CNS neoplastic cells draw attention to the virus’ oncolytic potential. Studies demonstrating the safety of using ZIKV for treating malignant CNS tumors, enabling the translation of this approach to clinical trials, are scarce in the literature. Here we developed a co-culture model of mature human cerebral organoids assembled with GBM, MED or ATRT tumor cells and used these assembloids to test ZIKV oncolytic effect, replication potential and preferential targeting between normal and cancer cells. Our hybrid co-culture models allowed the tracking of tumor cell growth and invasion in cerebral organoids. ZIKV replication and ensuing accumulation in the culture medium was higher in organoids co-cultured with tumor cells than in isolated control organoids without tumor cells. ZIKV infection led to a significant reduction in tumor cell proportion in organoids with GBM and MED cells, but not with ATRT. Tumoroids (3D cultures of tumor cells alone) were efficiently infected by ZIKV. Interestingly, ZIKV rapidly replicated in GBM, MED, and ATRT tumoroids reaching significantly higher viral RNA accumulation levels than co-cultures. Moreover, ZIKV infection reduced viable cells number in MED and ATRT tumoroids but not in GBM tumoroids. Altogether, our findings indicate that ZIKV has greater replication rates in aggressive CNS tumor cells than in normal human cells comprising cerebral organoids. However, such higher ZIKV replication in tumor cells does not necessarily parallels oncolytic effects, suggesting cellular intrinsic and extrinsic factors mediating tumor cell death by ZIKV.

Congenital Zika syndrome (CZS) is a set of birth defects caused by Zika virus (ZIKV) infection during pregnancy. Microcephaly is its main feature, but other brain abnormalities are found in CZS patients, such as ventriculomegaly, brain calcifications, and dysgenesis of the corpus callosum. Many studies have focused on microcephaly, but it remains unknown how ZIKV infection leads to callosal malformation. To tackle this issue, we infected mouse embryos in utero with a Brazilian ZIKV isolate and found that they were born with a reduction in callosal area and density of callosal neurons. ZIKV infection also causes a density reduction in PH3+ cells, intermediate progenitor cells, and SATB2+ neurons. Moreover, axonal tracing revealed that callosal axons are reduced and misrouted. Also, ZIKV-infected cultures show a reduction in callosal axon length. GFAP labeling showed that an in utero infection compromises glial cells responsible for midline axon guidance. In sum, we showed that ZIKV infection impairs critical steps of corpus callosum formation by disrupting not only neurogenesis, but also axon guidance and growth across the midline.

VAMP (Vesicle-associated membrane protein)-associated protein B and C (VAPB) has been widely studied in neurodegenerative diseases such as ALS, but little is known about its role in cancer. Medulloblastoma is a common brain malignancy in children and arises from undifferentiated cells during neuronal development. Therefore, medulloblastoma is an interesting model to investigate the possible relationship between VAPB and tumorigenesis. Here we demonstrate that high VAPB expression in medulloblastoma correlates with decreased overall patient survival. Consistent with this clinical correlation, we find that VAPB is required for normal proliferation rates of medulloblastoma cells in vitro and in vivo. Knockout of VAPB (VAPB^KO) delayed cell cycle progression. Furthermore, transcript levels of WNT-related proteins were decreased in the VAPB^KO. We conclude that VAPB is required for proliferation of medulloblastoma cells, thus revealing VAPB as a potential therapeutic target for medulloblastoma treatment.

Congenital Zika Syndrome (CZS) is a set of birth defects caused by Zika virus (ZIKV) infection during pregnancy. Microcephaly is its main feature, but other brain abnormalities are found in CZS patients, such as ventriculomegaly, brain calcifications, and dysgenesis of the corpus callosum. Many studies have focused on microcephaly, but it remains unknown how ZIKV infection leads to callosal malformation. To tackle this issue, we infected mouse embryos in utero with a Brazilian ZIKV isolate and found that they are born with a reduction in callosal area and density of callosal neurons. ZIKV infection also causes a density reduction of PH3+ cells, intermediate progenitor cells and SATB2+ neurons. Moreover, axonal tracing revealed that callosal axons are reduced and misrouted. Also, ZIKV infected cultures show a reduction of callosal axon length. GFAP labelling showed that in utero infection compromises glial cells responsible for midline axon guidance. The RNA-Seq data from infected brains identified downregulation of axon guidance and axonogenesis related genes. In sum, we showed that ZIKV infection impairs critical steps of corpus callosum formation by disrupting not only neurogenesis but also axon guidance and growth across the midline. Summary Statement: Zika virus infection during development impairs the formation of corpus callosum by disturbing axon guidance and growth of callosal neurons.