Deep Learning Reveals Cancer Metastasis and Therapeutic Antibody Targeting in the Entire Body.

Reliable detection of disseminated tumor cells and of the biodistribution of tumor-targeting therapeutic antibodies within the entire body has long been needed to better understand and treat cancer metastasis. Here, we developed an integrated pipeline for automated quantification of cancer metastases and therapeutic antibody targeting, named DeepMACT. First, we enhanced the fluorescent signal of cancer cells more than 100-fold by applying the vDISCO method to image metastasis in transparent mice. Second, we developed deep learning algorithms for automated quantification of metastases with an accuracy matching human expert manual annotation. Deep learning-based quantification in 5 different metastatic cancer models including breast, lung, and pancreatic cancer with distinct organotropisms allowed us to systematically analyze features such as size, shape, spatial distribution, and the degree to which metastases are targeted by a therapeutic monoclonal antibody in entire mice. DeepMACT can thus considerably improve the discovery of effective antibody-based therapeutics at the pre-clinical stage. VIDEO ABSTRACT.

Shrinkage-mediated imaging of entire organs and organisms using uDISCO.

Recent tissue-clearing approaches have become important alternatives to standard histology approaches. However, light scattering in thick tissues and the size restrictions on samples that can be imaged with standard light-sheet microscopy pose limitations for analyzing large samples such as an entire rodent body. We developed 'ultimate DISCO' (uDISCO) clearing to overcome these limitations in volumetric imaging. uDISCO preserves fluorescent proteins over months and renders intact organs and rodent bodies transparent while reducing their size up to 65%. We used uDISCO to image neuronal connections and vasculature from head to toe over 7 cm and to perform unbiased screening of transplanted stem cells within the entire body of adult mice. uDISCO is compatible with diverse labeling methods and archival human tissue, and it can readily be used in various biomedical applications to study organization of large organ systems throughout entire organisms.

The choroid plexus is a key cerebral invasion route for T cells after stroke.

Neuroinflammation contributes substantially to stroke pathophysiology. Cerebral invasion of peripheral leukocytes-particularly T cells-has been shown to be a key event promoting inflammatory tissue damage after stroke. While previous research has focused on the vascular invasion of T cells into the ischemic brain, the choroid plexus (ChP) as an alternative cerebral T-cell invasion route after stroke has not been investigated. We here report specific accumulation of T cells in the peri-infarct cortex and detection of T cells as the predominant population in the ipsilateral ChP in mice as well as in human post-stroke autopsy samples. T-cell migration from the ChP to the peri-infarct cortex was confirmed by in vivo cell tracking of photoactivated T cells. In turn, significantly less T cells invaded the ischemic brain after photothrombotic lesion of the ipsilateral ChP and in a stroke model encompassing ChP ischemia. We detected a gradient of CCR2 ligands as the potential driving force and characterized the neuroanatomical pathway for the intracerebral migration. In summary, our study demonstrates that the ChP is a key invasion route for post-stroke cerebral T-cell invasion and describes a CCR2-ligand gradient between cortex and ChP as the potential driving mechanism for this invasion route.

Selective plasticity of callosal neurons in the adult contralesional cortex following murine traumatic brain injury.

Traumatic brain injury (TBI) results in deficits that are often followed by recovery. The contralesional cortex can contribute to this process but how distinct contralesional neurons and circuits respond to injury remains to be determined. To unravel adaptations in the contralesional cortex, we used chronic in vivo two-photon imaging. We observed a general decrease in spine density with concomitant changes in spine dynamics over time. With retrograde co-labeling techniques, we showed that callosal neurons are uniquely affected by and responsive to TBI. To elucidate circuit connectivity, we used monosynaptic rabies tracing, clearing techniques and histology. We demonstrate that contralesional callosal neurons adapt their input circuitry by strengthening ipsilateral connections from pre-connected areas. Finally, functional in vivo two-photon imaging demonstrates that the restoration of pre-synaptic circuitry parallels the restoration of callosal activity patterns. Taken together our study thus delineates how callosal neurons structurally and functionally adapt following a contralateral murine TBI.

The effect of Pyrroloquinoline quinone and Resveratrol on the Survival and Regeneration of Cerebellar Granular Neurons.

The inability of central nervous system (CNS) to regenerate following traumatic brain injury (TBI) can be attributed to apoptotic cell death, inhibitory extrinsic environment, and the limited ability of neurons to regenerate. Thus, fostering the intrinsic regenerative potential and minimizing neuronal cell death could be a promising therapeutic approach. Pyrroloquinoline quinone (PQQ) was previously reported for its neuroprotective and regenerative potential on peripheral nerves. Here, we investigated the ability of PQQ to induce neurite re-growth in a wound healing model on cultured cerebellar granular neurons (CGNs), an integral part of cerebellar circuitry, as one of the most affected areas following TBI. The neuroprotective effect was also examined utilizing K+/FCS deprivation-induced apoptosis model in CGNs. Resveratrol (RVT), an effective promoter of neuroprotection and regeneration both centrally and peripherally was also investigated separately and in combination with PQQ to establish a possible synergistic effect. RVT (5 µM) and PQQ (0.5 µM) showed a tendency to promote neurite re-growth in the wound healing assay, however the effect observed was statistically insignificant. Higher concentrations of PQQ (1 and 2 µM) were found to be less effective. Resveratrol did not affect neurite length in CGNs culture; however, it did significantly increase the number of viable CGNs. For the neuroprotective effect; PQQ and RVT showed a significant increase in the survival of CGNs following K+/FCS deprivation of culture. Thus, both compounds showed a tendency to support neurite outgrowth in addition to a significant neuroprotective effect, but no synergistic effect was detected.

Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull-meninges connections.

Analysis of entire transparent rodent bodies after clearing could provide holistic biological information in health and disease, but reliable imaging and quantification of fluorescent protein signals deep inside the tissues has remained a challenge. Here, we developed vDISCO, a pressure-driven, nanobody-based whole-body immunolabeling technology to enhance the signal of fluorescent proteins by up to two orders of magnitude. This allowed us to image and quantify subcellular details through bones, skin and highly autofluorescent tissues of intact transparent mice. For the first time, we visualized whole-body neuronal projections in adult mice. We assessed CNS trauma effects in the whole body and found degeneration of peripheral nerve terminals in the torso. Furthermore, vDISCO revealed short vascular connections between skull marrow and brain meninges, which were filled with immune cells upon stroke. Thus, our new approach enables unbiased comprehensive studies of the interactions between the nervous system and the rest of the body.

Loss of TREM2 function increases amyloid seeding but reduces plaque-associated ApoE.

Coding variants in the triggering receptor expressed on myeloid cells 2 (TREM2) are associated with late-onset Alzheimer's disease (AD). We demonstrate that amyloid plaque seeding is increased in the absence of functional Trem2. Increased seeding is accompanied by decreased microglial clustering around newly seeded plaques and reduced plaque-associated apolipoprotein E (ApoE). Reduced ApoE deposition in plaques is also observed in brains of AD patients carrying TREM2 coding variants. Proteomic analyses and microglia depletion experiments revealed microglia as one origin of plaque-associated ApoE. Longitudinal amyloid small animal positron emission tomography demonstrates accelerated amyloidogenesis in Trem2 loss-of-function mutants at early stages, which progressed at a lower rate with aging. These findings suggest that in the absence of functional Trem2, early amyloidogenesis is accelerated due to reduced phagocytic clearance of amyloid seeds despite reduced plaque-associated ApoE.