Arf1-KO Induces Anti-Tumour Response with Protective Vaccination

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By IMPC

Published 27th April 2020

A study investigating the role of Arf1 shows promising results for new therapeutic strategies involving DAMP-mediated anti-tumour immunity to target cancer stem cells. This method of inducting an anti-tumour response could be another frontier in anti-tumour immune therapy.

“[This could be] the first demonstration that endogenous metabolic stress elicits anti-tumour immune responses by inducing DAMPs in rodent tumor models.”

Cancer stem cells (CSCs) are found in tumours and, like other stem cell types, have the ability to give rise to many different cell types. Their affinity for self-renewal, differentiation, tumourigenicity and their possible resistance to traditional treatments like chemotherapy suggests they could be responsible for treatment resistance, tumour metastasis, reoccurrence, evasion and patient death. CSCs, therefore, prove a good target for future tumour therapies.

CSCs have a unique metabolism. Leukaemia stem cells have been shown to rely on amino acids for oxidative phosphorylation and some CSC-enriched disseminated tumour cells obtain energy from fatty acids. The researchers’ previous work with drosophila found that the COPI/Arf1-mediated lipolysis pathway selectively sustains and transforms stem cells and that targeting this pathway by knocking out Arf1 kills stems cells via necrosis.

In this study, the researchers investigated the Arf1-mediated lipid metabolism pathway’s role in sustaining CSCs in mice and how disrupting this pathway would affect tumour growth and number. IMPC Arf1 embryonic stem cells were used in this study.

Does the Arf1 Lipolysis Pathway Sustain CSCs?

The researchers used the Lgr5-CreERT2/Apcf/f (Lgr5/Apc) mouse model, one known to be a good model for CSCs, and knocked out Arf1 to produce Lgr5-CreERT2/Arf1f/f/Apcf/f mice. They found a dramatic reduction in stem cell tumour numbers and a significant increase in lifespan for the Lgr4/Arf1/Apc mice. After examining intestinal cells, they found abnormal mitochondria, poor cristae, vacuoles, degeneration and necrosis.

Please see source scientific journal article for figure caption.

They then took mouse colon cancer CT26 cells and human liver cancer Huh-7 cells and treated them with Arf1 inhibitors, BFA and GCA. The treatment dramatically increased lipid droplet accumulation in both cell types.

Deletion of Arf1 from the ventral foregut endoderm with Foxa3-Cre resulted in significantly underdeveloped livers and lethality at embryonic day 15.5. Further study found that it also caused a significant reduction in hepatoblast markers Hnf4α and Hnf6, lipid droplet accumulation and necrotic death of hepatoblasts.

“The Arf1-regulated lipolysis pathway selectively sustains stem cells, progenitors and cells enriched with CSCs in mice and that disrupting the pathway in these cells results in lipid droplet accumulation, mitochondrial defects and cell necrosis.”

Arf1-KO’s Effect on Intestinal CSCs

Arf1 depleted mice showed accumulation of CD3+, CD8+ and CD4+, evidence of a T-cell immune response, with dendritic cells (DCs) and inflammatory DCs also being significantly increased. Upon examining the population of lamina propria lymphocytes within the GI tract, they found that certain T-cells (CD8/4+ with and without IFN-γ+) were all significantly increased. These changes were not seen in other areas within the GI tract nor were these changes observed in other key immune cell sites, including the spleen, draining lymph nodes and inguinal lymph nodes.

T-cell-related chemokines (CCL5, CXCL10 and CCL22) were upregulated in the Lgr5/Arf1/Apc model. Real-time PCR showed elevated expressions of IFN-γ, perforin, granzyme A and B and IL-1β. The accumulated evidence suggested that:

“knocking out Arf1 in Lgr5+ stem cells triggers T cell infiltration and activation, leading to CSC death and prolonged survival of Lgr5/Arf1/Apc mice.”

Arf1-KO’s Effect on Liver CSCs

The researchers induced liver tumours using the Tet-o-MYC/LAP-tTA system before adding the Arf1 inhibitors GCA or BFA to mouse food, dramatically reducing the number of liver tumours and extending the lifespan of the mice. Selective depletion of Arf1 in Axin2+ liver cells or CSCs also produced this effect.

DCs, B cells and both CD4+ and CD8+ T-cells were significantly increased in the liver, as were many T-cell-related cytokines (CXCL10, CXCL11 and CCL22.) Real-time PCR showed elevated expression levels for the same molecules as in the intestine. Real-time PCR also, however, showed a significant decrease in PD-L1 expression in several mouse models and cell types.

“Arf1 inhibition may down-regulate PD-L1 through reducing the AP-1/C-Jun pathway…PD-L1 reduction could be partially responsible for the activation of infiltrated T cells that enhances the anti-tumour effect of Arf1 ablation”

Defining the Effect: T-cell Activation, DC Infiltration and DAMPs

In order to confirm that the effect relied on an adaptive immune response, they used anti-CD4 and anti-CD8 antibodies to deplete T-cells. In response, tumour numbers dramatically increased and lifespans were shortened. They then additionally knocked out either Rag1 or IFN-γ in the Lgr5/Arf1/Apc mice. Both caused survival times to significantly decrease. They concluded that the anti-tumour effects were due to an induction of a T-cell-dependant immune response.

Knocking out Arf1 had an effect on other aspects of the immune system. They found Arf1 inhibition may also induce inflammasome-mediated cell necrosis/pyroptosis. Arf1 inhibition or ablation also induced key DAMPS like Calr, HMGBI, ERp46 and LAMP1, as well as ER-stress markers. It was concluded that Arf1 inhibition triggers ER stress and, as a result, induces DAMPS and DC infiltration, enhancing the T-cell infiltration.


a–b Huh-7 cells were treated with control DMSO or GCA and then injected into athymic nude mice…Calr, HMGB1, ERp46, and the lysosome protein LAMP1 were dramatically induced in the GCA-treated tumours (a and b). Nuclear HMGB1 moved to the extracellular space (b).”

Through tests with a series of inhibitors, including the ATPase inhibitor ARL, it was concluded that the anti-tumour effect worked via ATP and HMGBI. Arf1 knockdown was not directly cytotoxic to tumour cells and, together with the above conclusions, the anti-tumour effect was DCs-ATP-IFN-γ-mediated T-cell immunity. Knockdown also prevented tumour metastasis.

Using Arf1-KO Cells for Vaccination

Vaccination with knockout Arf1 cells also proved successful in protecting animals from developing tumours. The vaccination was effective not only against melanoma but also against a variety of histologic types like colon cancer and breast carcinomas. Arf1 inhibition triggered a localised T-cell immune response that then attacked cancer throughout the body.

Please see source scientific journal article for figure caption.

Simultaneous inhibition of T-cell checkpoint receptor PD-1 with Arf1 ablation had a synergistic effect and further reduced tumour numbers.

Upon analysing public datasets on human cancer, the researchers found Arf1 was amplified or overexpressed in the majority of cancer types examined. The cases with lower expression levels of Arf1 had significantly better survival probabilities for a number of cancer types, suggesting Arf1 is a negative prognostic factor.

How it All Happens

The researchers proposed a model:

“a small number of active anti-CSC T cells penetrate the tumor and attack…; as a result…the CTLs produce cytokines and re-stimulate the local environment to convert an immunosuppressive to an immunostimulatory environment;…[reawakening] the pre-existing inactive anti-tumor T cells [and recruiting] new anti-tumor T cells; these additional T cells are directed against tumour antigens other than the DAMPs to amplify the effects for destroying the bulk tumors; these active T cells may then migrate to other tumor sites and become memory T cells to produce widespread and durable antitumor effects.”

“Proposed model depicting how Arf1 knockdown promotes metabolic stress, the induction of DAMPs, and immune cell infiltration and activation to attack tumours”

If results like the above are reproducible in humans, DAMP-mediated anti-tumour immune therapy could be a breakthrough for the treatment of reoccurring tumours, particularly those with high Arf1 expression. When used in conjunction with other already effective treatments or with the addition of successful checkpoint blockades, this method could prove even more effective.

Source:

Wang, G., Xu, J., Zhao, J. et al. Arf1-mediated lipid metabolism sustains cancer cells and its ablation induces anti-tumor immune responses in mice. Nat Commun11, 220 (2020). https://doi.org/10.1038/s41467-019-14046-9

Scientists at the Champalimaud Centre for the Unknown and the Instituto de Medicina Molecular, in Lisbon, Portugal, have discovered that neurons located at mucosal tissues can immediately detect an infection in the organism, promptly producing a substance that acts as an “adrenaline rush” for immune cells. Under the effect of this signal, immune cells rapidly become poised to fight the infection and repair the damage caused to surrounding tissues. These results have been published in the journal Nature. The research utilised the KOMP Repository for obtaining Nmur1 deleted mouse strains.

Most neurons in the body are located in the brain and its vicinity – the central nervous system -, with neurons projecting their axons to every tissue in the organism by way of the spinal cord. In turn, glial cells are neuron satellites ensuring the cohesion of the nervous tissue. Nevertheless, throughout the body there is a very abundant number of peripheral nervous cells. These are so numerous in the gut that they have collectively been dubbed “the second brain”.

Innate lymphocytes (in green) surround the gut (in red). Credit: Henrique Veiga-Fernandes

What do these peripheral nervous cells do? Experts are just beginning to understand that they are in fact extremely important for the organism to be able to mount adequate immune responses and preserve health.

In 2016, Henrique Veiga-Fernandes and his colleagues (then at the Institute for Molecular Medicine, in Lisbon) published, also in Nature, a study where they showed that glial cells in the gut can stimulate a type of immune cells, called ILC3, to produce substances against bacterial infections.

These immune cells that are being studied by Veiga-Fernandes – collectively called “innate lymphoid cells”, or ILC -, are also very special. We are born with them; they are not produced in response to an immunization, for instance through vaccination. “ILCs were discovered very recently, in 2010, but they are very ancient in evolutionary terms. Even lampreys have them!”, says Veiga-Fernandes. Lampreys belong to a very old animal lineage.

There are several types of these innate lymphocytes (white cells). In their 2016 study, the team had analyzed the behavior of ILC3s in the gut – and their “dialog” with their glial cell neighbours. In the new study, also led by Veiga-Fernandes, they focused on another type of innate lymphoid cells: ILC2s.

ILC2s produce substances that are essential to immune responses against parasites, such as worms. “These cells are normally abundant at barrier sites, such as the gut, lungs and skin”, which serve as physical forteresses to the body”, Veiga-Fernandes explains.

Now, the team showed that these immune cells would not be able to develop their protective actions against infections without establishing a “dialog” with neurons residing at those sites.

The study brings “two big novelties”, says Veiga-Fernandes. The first, he explains, “is that neurons define the immune cells’ function. Nobody could have imagined that the nervous system coordinates, commands and controls the immune response throughout the whole organism.” Second, he adds, “it’s one of the fastest and most powerful immune reactions we have ever seen”. Comparatively, the newly discovered neuronal stimulus induces an immune response in a few minutes, while the immune response following vaccination typically takes several weeks to mount.

How did the scientists discover this neuro-immune “tandem”? “What happened was that we observed, in high-resolution microphotographs of the lungs and gut of mice, that ILC2s were placed along the axons of neurons residing in these mucosa, a bit like pearls on a string”, replies Veiga-Fernandes. “So we asked ourselves if these two distinct tissues could productively ‘talk’ to each other.”

To test this hypothesis, the team started by analyzing the whole genome of a series of immune cells – ILC1s, ILC2s, ILC3s, T-cells, etc. -, “searching for genes that code molecules that may act as receivers of neuronal signals”, says Veiga-Fernandes. They found that only ILC2s possessed a defined “receptor” (membrane molecules that act as antennae) for nervous signals.

Notably, the authors discovered that ILC2s have receptors to a neuronal messenger called neuromedin U (NMU). Since neurons are the only cells that produce abundant levels of NMU, this indicated that only neurons could be sending this signal to ILC2s.

Later, they used a rodent parasite, Nippostrongylus brasiliensis (a sort of hookworm) to infect “normal” control mice and mutant mice whose ILC2s had been stripped of their NMU receptors. In the first group of animals, the innate immune cells immediately triggered a response to neutralize the parasite and repair damaged tissue. In the second group, the mice were unable to fight the infection and the damage caused by the parasite – including the internal bleeding of the lungs due to N. brasiliensis.

The researchers also showed that neurons are able to detect the products secreted by parasites that infect the organism – and that, when this happens, they rapidly produce NMU. In turn, NMU acts vigorously on ILC2s, thus generating a protective response in a few minutes.

Could these results be extrapolated to humans? “Maybe. In humans, ILC2s also have NMU receptors”, replies Veiga-Fernandes. “But we are still very far from understanding how we could safely use this neuro-immunological ‘bomb’; for now, we are at the fundamental research level”, he adds.

Research Article: Neuronal regulation of type 2 innate lymphoid cells via neuromedin U

Nerve cells in the gut play a crucial role in the body’s ability to marshal an immune response to infection, according to a new study from Weill Cornell Medicine scientists.

The study, published in Nature, shows that the immune system and nervous system have co-evolved to respond to infectious threats. This means that scientists looking for ways to treat diseases like inflammatory bowel disease or asthma that involve an excessive immune system response may also have to address the nervous system’s role.

“The immune system and neuronal system don’t act independently,” said senior author Dr. David Artis, director of the Jill Roberts Institute for Research in Inflammatory Bowel Disease and the Michael Kors Professor of Immunology at Weill Cornell Medicine. “They are working together.”

“These guys are dancing like a tango,” Dr. Klose said. The lining of the gut is home to many immune system cells, which serve as a defense against parasites and other infections. It is also loaded with nerve cells. Lead author Dr. Christoph Klose, a postdoctoral associate at the Roberts Institute, found that immune system cells in the gut, called group 2 innate lymphoid cells (ILC2s), are intertwined with nerve cells called cholinergic neurons.

The intestinal barrier. Color-enhanced image of the intestinal lining, which serves as a barrier and limits dissemination of the microbiota. Credit: Greg Sonnenberg and David Artis, Weill Cornell Medicine.

The cells’ close proximity led the researchers to wonder if they may be communicating. That’s when they discovered that the ILC2 cells had a receptor for a protein called neuromedin U (NMU), which acts as a messenger for the nerve cells. In laboratory experiments, the investigators found that exposing ILC2 cells to NMU causes the ILC2 cells to rapidly multiply and secrete chemicals called cytokines that may help trigger an immune response or cause inflammation.

Administering NMU to mice infected with a gut parasite triggered inflammation and a powerful immune response that helped the mice more quickly expel the parasites. Conversely, mice genetically engineered to lack receptors for NMU were more susceptible to the parasites, allowing them to multiply rapidly in the rodents’ gut. The study shows that the NMU-producing nerve cells help prime the ILC2 cells, enabling them to rapidly and effectively respond to infection.

“Where we are most excited is thinking about multiple chronic inflammatory diseases that might be related to this neuronal-immune axis and where we might be able to intervene,” Dr. Artis said. The findings may have important implications for scientists studying inflammatory diseases, including asthma, food allergies and inflammatory bowel disease (IBD). Dr. Artis said it was too soon to say whether NMU itself or its receptors could be treatment targets, but he said studying these pathways might lead to potential new therapies for these diseases.

 

Research article: The neuropeptide neuromedin U stimulates innate lymphoid cells and type 2 inflammation

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By IMPC

Published 6th July 2018

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