A protocol to measure lysosomal Zn2+ release through a genetically encoded Zn2+ indicator.

Intracellular vesicles such as lysosomes contain micromolar to millimolar concentrations of Zn2+, and disturbing lysosomal Zn2+ homeostasis via lysosomal Zn2+ release leads to mitochondria damage and consequent lytic cell death. Methods have been developed to image cellular Zn2+ dynamics. Here, we present a protocol using GZnP3, a genetically encoded fluorescent Zn2+ indicator, to assess lysosomal Zn2+ release in cultured cells by fluorescence microscopy imaging. For complete details on the use and execution of this protocol, please refer to Du et al. (2021) or Minckley et al. (2019).

Parkinson's disease-risk protein TMEM175 is a proton-activated proton channel in lysosomes.

Lysosomes require an acidic lumen between pH 4.5 and 5.0 for effective digestion of macromolecules. This pH optimum is maintained by proton influx produced by the V-ATPase and efflux through an unidentified "H+ leak" pathway. Here we show that TMEM175, a genetic risk factor for Parkinson's disease (PD), mediates the lysosomal H+ leak by acting as a proton-activated, proton-selective channel on the lysosomal membrane (LyPAP). Acidification beyond the normal range potently activated LyPAP to terminate further acidification of lysosomes. An endogenous polyunsaturated fatty acid and synthetic agonists also activated TMEM175 to trigger lysosomal proton release. TMEM175 deficiency caused lysosomal over-acidification, impaired proteolytic activity, and facilitated α-synuclein aggregation in vivo. Mutational and pH normalization analyses indicated that the channel's H+ conductance is essential for normal lysosome function. Thus, modulation of LyPAP by cellular cues may dynamically tune the pH optima of endosomes and lysosomes to regulate lysosomal degradation and PD pathology.

Lysosomal Zn2+ release triggers rapid, mitochondria-mediated, non-apoptotic cell death in metastatic melanoma.

During tumor progression, lysosome function is often maladaptively upregulated to match the high energy demand required for cancer cell hyper-proliferation and invasion. Here, we report that mucolipin TRP channel 1 (TRPML1), a lysosomal Ca2+ and Zn2+ release channel that regulates multiple aspects of lysosome function, is dramatically upregulated in metastatic melanoma cells compared with normal cells. TRPML-specific synthetic agonists (ML-SAs) are sufficient to induce rapid (within hours) lysosomal Zn2+-dependent necrotic cell death in metastatic melanoma cells while completely sparing normal cells. ML-SA-caused mitochondria swelling and dysfunction lead to cellular ATP depletion. While pharmacological inhibition or genetic silencing of TRPML1 in metastatic melanoma cells prevents such cell death, overexpression of TRPML1 in normal cells confers ML-SA vulnerability. In the melanoma mouse models, ML-SAs exhibit potent in vivo efficacy of suppressing tumor progression. Hence, targeting maladaptively upregulated lysosome machinery can selectively eradicate metastatic tumor cells in vitro and in vivo.

Anti-EGFR VHH-armed death receptor ligand-engineered allogeneic stem cells have therapeutic efficacy in diverse brain metastatic breast cancers.

Basal-like breast cancer (BLBC) shows brain metastatic (BM) capability and overexpresses EGFR and death-receptors 4/5 (DR4/5); however, the anatomical location of BM prohibits efficient drug-delivery to these targetable markers. In this study, we developed BLBC-BM mouse models featuring different patterns of BMs and explored the versatility of estem cell (SC)-mediated bi-functional EGFR and DR4/5-targeted treatment in these models. Most BLBC lines demonstrated a high sensitivity to EGFR and DR4/5 bi-targeting therapeutic protein, EVDRL [anti-EGFR VHH (EV) fused to DR ligand (DRL)]. Functional analyses using inhibitors and CRISPR-Cas9 knockouts revealed that the EV domain facilitated in augmenting DR4/5-DRL binding and enhancing DRL-induced apoptosis. EVDRL secreting stem cells alleviated tumor-burden and significantly increased survival in mouse models of residual-tumor after macrometastasis resection, perivascular niche micrometastasis, and leptomeningeal metastasis. This study reports mechanism based simultaneous targeting of EGFR and DR4/5 in BLBC and defines a new treatment paradigm for treatment of BM.

Next-generation immunotherapies for brain metastatic cancers.

Patients with extracranial tumors, like lung, breast, and skin cancers, often develop brain metastases (BM) during the course of their diseases and BM commonly represent the terminal stage of cancer progression. Recent insights in the immune biology of BM and the increasing focus of immunotherapy as a therapeutic option for cancer has prompted testing of promising biological immunotherapies, including immune cell-targeting, virotherapy, vaccines, and different cell-based therapies. Here, we review the pathobiology of BM progression and evaluate the potential of next-generation immunotherapies for BM tumors. We also provide future perspectives on the development and implementation of such therapies for brain metastatic cancer patients.

CRISPR-enhanced engineering of therapy-sensitive cancer cells for self-targeting of primary and metastatic tumors.

Tumor cells engineered to express therapeutic agents have shown promise to treat cancer. However, their potential to target cell surface receptors specific to the tumor site and their posttreatment fate have not been explored. We created therapeutic tumor cells expressing ligands specific to primary and recurrent tumor sites (receptor self-targeted tumor cells) and extensively characterized two different approaches using (i) therapy-resistant cancer cells, engineered with secretable death receptor-targeting ligands for "off-the-shelf" therapy in primary tumor settings, and (ii) therapy-sensitive cancer cells, which were CRISPR-engineered to knock out therapy-specific cell surface receptors before engineering with receptor self-targeted ligands and reapplied in autologous models of recurrent or metastatic disease. We show that both approaches allow high expression of targeted ligands that induce tumor cell killing and translate into marked survival benefits in mouse models of multiple cancer types. Safe elimination of therapeutic cancer cells after treatment was achieved by co-engineering with a prodrug-converting suicide system, which also allowed for real-time in vivo positron emission tomography imaging of therapeutic tumor cell fate. This study demonstrates self-tumor tropism of engineered cancer cells and their therapeutic potential when engineered with receptor self-targeted molecules, and it establishes a roadmap toward a safe clinical translation for different cancer types in primary, recurrent, and metastatic settings.

Stem cell-released oncolytic herpes simplex virus has therapeutic efficacy in brain metastatic melanomas.

The recent Food and Drug Administration approval of immunogenic oncolytic virus (OV) has opened a new era in the treatment of advanced melanoma; however, approximately 50% of patients with melanoma develop brain metastasis, and currently there are no beneficial treatment options for such patients. To model the progression of metastases seen in patients and to overcome the hurdles of systemic delivery of OV, we developed melanoma brain metastasis models in immunocompromised and immunocompetent mice, and tested the fate and efficacy of oncolytic herpes simplex virus (oHSV)-armed mesenchymal stem cells (MSCs). Using brain-seeking patient-derived melanoma cells and real-time in vivo imaging, we show a widespread distribution of micrometastases and macrometastases in the brain, recapitulating the progression of multifoci metastases seen in patients. We armed MSCs with different oHSV variants (MSC-oHSV) and found that intracarotid administration of MSC-oHSV, but not of purified oHSV alone, effectively tracks metastatic tumor lesions and significantly prolongs the survival of brain tumor-bearing mice. In a syngeneic model of melanoma brain metastasis, a combination of MSC-oHSV and PD-L1 blockade increases IFNγ-producing CD8+ tumor-infiltrating T lymphocytes and results in a profound extension of the median survival of treated animals. This study thus demonstrates the utility of MSCs as OV carriers to disseminated brain lesions, and provides a clinically applicable therapeutic platform to target melanoma brain metastasis.

Targeting breast to brain metastatic tumours with death receptor ligand expressing therapeutic stem cells.

Characterizing clinically relevant brain metastasis models and assessing the therapeutic efficacy in such models are fundamental for the development of novel therapies for metastatic brain cancers. In this study, we have developed an in vivo imageable breast-to-brain metastasis mouse model. Using real time in vivo imaging and subsequent composite fluorescence imaging, we show a widespread distribution of micro- and macro-metastasis in different stages of metastatic progression. We also show extravasation of tumour cells and the close association of tumour cells with blood vessels in the brain thus mimicking the multi-foci metastases observed in the clinics. Next, we explored the ability of engineered adult stem cells to track metastatic deposits in this model and show that engineered stem cells either implanted or injected via circulation efficiently home to metastatic tumour deposits in the brain. Based on the recent findings that metastatic tumour cells adopt unique mechanisms of evading apoptosis to successfully colonize in the brain, we reasoned that TNF receptor superfamily member 10A/10B apoptosis-inducing ligand (TRAIL) based pro-apoptotic therapies that induce death receptor signalling within the metastatic tumour cells might be a favourable therapeutic approach. We engineered stem cells to express a tumour selective, potent and secretable variant of a TRAIL, S-TRAIL, and show that these cells significantly suppressed metastatic tumour growth and prolonged the survival of mice bearing metastatic breast tumours. Furthermore, the incorporation of pro-drug converting enzyme, herpes simplex virus thymidine kinase, into therapeutic S-TRAIL secreting stem cells allowed their eradication post-tumour treatment. These studies are the first of their kind that provide insight into targeting brain metastasis with stem-cell mediated delivery of pro-apoptotic ligands and have important clinical implications.

Targeting c-Met receptor overcomes TRAIL-resistance in brain tumors.

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induced apoptosis specifically in tumor cells. However, with approximately half of all known tumor lines being resistant to TRAIL, the identification of TRAIL sensitizers and their mechanism of action become critical to broadly use TRAIL as a therapeutic agent. In this study, we explored whether c-Met protein contributes to TRAIL sensitivity. We found a direct correlation between the c-Met expression level and TRAIL resistance. We show that the knock down c-Met protein, but not inhibition, sensitized brain tumor cells to TRAIL-mediated apoptosis by interrupting the interaction between c-Met and TRAIL cognate death receptor (DR) 5. This interruption greatly induces the formation of death-inducing signaling complex (DISC) and subsequent downstream apoptosis signaling. Using intracranially implanted brain tumor cells and stem cell (SC) lines engineered with different combinations of fluorescent and bioluminescent proteins, we show that SC expressing a potent and secretable TRAIL (S-TRAIL) have a significant anti-tumor effect in mice bearing c-Met knock down of TRAIL-resistant brain tumors. To our best knowledge, this is the first study that demonstrates c-Met contributes to TRAIL sensitivity of brain tumor cells and has implications for developing effective therapies for brain tumor patients.

TRPC6 inhibited NMDA receptor activities and protected neurons from ischemic excitotoxicity.

Excitotoxicity induced by NMDA receptor-mediated intracellular Ca(2+) ([Ca(2+) ](i)) overload is a major cause of delayed neuronal death in cerebral ischemia. Transient receptor potential canonical (TRPC) 6 protects neurons from ischemic brain damage. However, the mechanisms by which TRPC6 protects neurons are largely unknown. Here, we reported that TRPC6 suppressed the [Ca(2+)](i) elevation induced by NMDA and protected neurons from excitotoxicity. Over-expressing or down-regulating TRPC6 suppressed or aggravated Ca(2+) overload under excitotoxicity, respectively. TRPC6 protected cultured neurons from damage caused by NMDA toxicity or oxygen glucose deprivation (OGD). Moreover, the infarct volume in TRPC6 transgenic (Tg) mice was smaller than that in wild-type (WT) littermates. The TRPC6 Tg mice had better behavior performance and lower mortality than their WT littermates. Thus, TRPC6 inhibited NMDA receptor-triggered neurotoxicity and protected neurons from ischemic brain damage. Increase in TRPC6 activity could be a potential strategy for stroke prevention and therapy.

Inhibition of TRPC6 degradation suppresses ischemic brain damage in rats.

Brain injury after focal cerebral ischemia, the most common cause of stroke, develops from a series of pathological processes, including excitotoxicity, inflammation, and apoptosis. While NMDA receptors have been implicated in excitotoxicity, attempts to prevent ischemic brain damage by blocking NMDA receptors have been disappointing. Disruption of neuroprotective pathways may be another avenue responsible for ischemic damage, and thus preservation of neuronal survival may be important for prevention of ischemic brain injury. Here, we report that suppression of proteolytic degradation of transient receptor potential canonical 6 (TRPC6) prevented ischemic neuronal cell death in a rat model of stroke. The TRPC6 protein level in neurons was greatly reduced in ischemia via NMDA receptor-dependent calpain proteolysis of the N-terminal domain of TRPC6 at Lys¹6. This downregulation was specific for TRPC6 and preceded neuronal death. In a rat model of ischemia, activating TRPC6 prevented neuronal death, while blocking TRPC6 increased sensitivity to ischemia. A fusion peptide derived from the calpain cleavage site in TRPC6 inhibited degradation of TRPC6, reduced infarct size, and improved behavioral performance measures via the cAMP response element-binding protein (CREB) signaling pathway. Thus, TRPC6 proteolysis contributed to ischemic neuronal cell death, and suppression of its degradation preserved neuronal survival and prevented ischemic brain damage.

Functional roles of TRPC channels in the developing brain.

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable, nonselective cation channels formed by homomeric or heteromeric complexes of TRPC proteins that contain six transmembrane domains. These channels can be activated through a phospholipase-C-dependent mechanism, making them sensors for environmental cues. Their expression begins early in embryonic days and remains in adulthood. These channels have important roles in the processes of neuronal development, including neural stem cell proliferation, cerebellar granule cell survival, axon path finding, neuronal morphogenesis, and synaptogenesis. In this review, we will discuss functional implications of TRPC channels during brain development.

Critical role of TRPC6 channels in the formation of excitatory synapses.

The transient receptor potential canonical (TRPC) channels are Ca2+-permeable, nonselective cation channels with different biological functions, but their roles in brain are largely unknown. Here we report that TRPC6 was localized to excitatory synapses and promoted their formation via a CaMKIV-CREB-dependent pathway. TRPC6 transgenic mice showed enhancement in spine formation, and spatial learning and memory in Morris water maze. These results reveal a previously unknown role of TRPC6 in synaptic and behavioral plasticity.

TRPC6 channels promote dendritic growth via the CaMKIV-CREB pathway.

The canonical transient receptor potential channels (TRPCs) are Ca(2+)-permeable nonselective cation channels with various physiological functions. Here, we report that TRPC6, a member of the TRPC family, promotes hippocampal neuron dendritic growth. The peak expression of TRPC6 in rat hippocampus was between postnatal day 7 and 14, a period known to be important for maximal dendritic growth. Overexpression of TRPC6 increased phosphorylation of Ca(2+)/calmodulin-dependent kinase IV (CaMKIV) and cAMP-response-element binding protein (CREB) and promoted dendritic growth in hippocampal cultures. Downregulation of TRPC6 by short hairpin RNA interference against TRPC6 suppressed phosphorylation of both CaMKIV and CREB and impaired dendritic growth. Expressing a dominant-negative form of CaMKIV or CREB blocked the TRPC6-induced dendritic growth. Furthermore, inhibition of Ca(2+) influx suppressed the TRPC6 effect on dendritic growth. Finally, in TRPC6 transgenic mice, the phosphorylation of CaMKIV and CREB was enhanced and the dendritic growth was also increased. In conclusion, TRPC6 promoted dendritic growth via the CaMKIV-CREB pathway. Our results thus revealed a novel role of TRPC6 during the development of the central nervous system (CNS).