Evaluating CAR NK Immunotherapy in Patient-Derived Colorectal Organoids

In recent years, great advances have been made in the field of immunotherapy to treat cancer. One of the most promising treatments involves engineering immune cells to express chimeric antigen receptors (CAR). These receptors are carefully designed to recognize antigens expressed on the surface of tumor cells. Once the target is recognized, the CAR-engineered immune cells can attack and kill the tumor cells. CAR T cells have been successfully used to treat certain blood cancers—three CAR T therapies for lymphoma and leukemia have gained US FDA approval. In these cases, T cells were taken from individual patients, grown and genetically-altered in the lab, then reintroduced into the same patient. Continue reading “Evaluating CAR NK Immunotherapy in Patient-Derived Colorectal Organoids”

How Gut Microbes Affect Our Brain

coliform bacteriaThink about the last time you gave a presentation. The feeling of having “butterflies in your stomach”. Or when you meet someone for the first time, that “gut feeling” of whether you two will get along. In our day-to-day lives, we often associate what happens in the gut with what goes on in our brain. In fact, scientific evidence suggests that our gut and our brain frequently communicate—through gut microbes. Apparently, the existence of trillions of bacteria and eukaryotes in our gut is not only crucial for our physical health, they may also be important for our mental health.

Continue reading “How Gut Microbes Affect Our Brain”

How Cheese Helped Us Understand Microbial Interactions

In almost every environment on earth, such as soil, human skin and gut, there lives a whole community of microbes—sometimes up to hundreds of species. It may seem like they all flourish in peace. But just like you may have friendly or hostile interactions with your neighbors, the different bacterial species interact in various ways. They may cooperate, compete or, sometimes, even kill each other. The interaction is complicated, and scientists have struggled to understand the nature of these microbiome interactions. How do microbiomes assemble and maintain stability? How do the interactions among different species affect gene expression?

Continue reading “How Cheese Helped Us Understand Microbial Interactions”

4 Things We Recently Learned About Cats

We know a lot about cats. We know that they’re adorable, they make us happy and they can survive a fall from a 32-story building. But apparently, there’s still a lot we don’t know about them. Here are four things we only recently discovered about cats.

1. Sharp, tiny “papillae” on cat tongues help deep-clean their fur.

Cats spend much of their time licking themselves. On the surface of their tongue are hundreds of tiny claw-shaped spines called papillae, each the size of half a grain of rice. Recently, a study published in PNAS revealed the purpose of these structures. The authors found that the tips of the papillae create a U-shaped cavity (imagine a coffee straw cut in half). The cavity holds saliva and helps move it down through the fur to the cat’s skin, allowing deeper cleaning. “Why should I care,” you ask? With these results, the authors created a brush that mimics a cat’s tongue. It tugged less and was easier to clean than a normal brush. The new design could be used to distribute cleaning solutions into carpets. Or, remove allergens from cat fur.

2. Feral cats aren’t great at controlling city rat population.

Many people assume that feral cats hunt and kill rats, helping control city rat population. However, a recent study showed that they’re not doing a great job. The researchers set up cameras around a rat colony in New York City and recorded the behaviors of feral cats that came near. A lucky graduate student watched all 306 videos and tallied cat behaviors such as walking, stalking and chasing. Over 79 days, only three times did a cat actively hunt a rat; and of the two cases in which the hunt was a success, the rats were relatively small in size. The presence of the cats did, however, cause the rats to hide more. Bottom line, let’s not release cats to control rat population.

3. Male cats tend to be left-pawed, and females right-pawed.

Just like humans can be left- or right-handed, cats also prefer using one of their paws over the other. The scientific term for this is called laterality, and it has been examined in many animals including rats, frogs, primates and whales. In this recent study, the paw preference of 44 pet cats were examined in their own homes. They found that most cats showed laterality when reaching for food (73%), stepping down stairs (70%) or stepping over their litter box (66%). While 90% of humans are right-handed, laterality in cats is much more equal—roughly half are left-biased and half right-biased. Surprisingly, the researchers also discovered that male cats are much more likely to prefer their left paw, while females prefer their right paw. The reason is unclear, but it may hint at the underlying differences of the male and female brains.

4. Cats domesticated themselves over thousands of years without much genetic change.

Humans can’t tell cats what to do—they do what they want, when they want. If you’ve ever had a cat, you know it’s true. In fact, this statement also applies to how they were domesticated. In a comprehensive study, researchers examined ancient DNA from more than 200 cats spanning the last 9,000 years and found that the modern domestic cat comes from two main lineages: one from southeast Asian and the other from Egypt. Cats likely began hanging out with humans when our ancestors began farming. With an abundance of rodents that fed on crops, cats voluntarily stayed close and slowly domesticated on their own. The genetic makeup of domesticated cats hasn’t changed much over thousands of years, except the appearance of striped or blotched tabby coat markings. And why would they change? They’re perfect just the way they are.

Dear Tech Serv, Thank You!

It’s that time of year again. Time to be thankful and show gratitude for those special people in your life. The undergrad who does the dishes, the labmate who shares their buffers when yours runs out, the collaborator that sends you data on a Saturday… Take a moment this week to say thank you, or send them an email to show your appreciation.

Today, we want to thank our Technical Services team. They work hard to help researchers choose the right assay for their needs, understand results and troubleshoot technical problems. They strive to provide the best service for those in need. Many on the receiving end have sent thankful messages:

“I deeply appreciate the help you have been and the email you just sent. I think with the information here, I may have sorted out an issue that has plagued our lab for the past few months.”

“Cannot tell you how grateful I am–you’ve been a tremendous help.”

“You are super sharp and caught critical errors in my protocol (the calculation and dilution errors you referenced below). While few of my colleagues run kinase assays, I did consult 6 of them, and none caught the errors you did. You’re clearly an expert and I truly appreciate how you’ve tailored everything for my ‘beginner’ level.”

“Wow, I cannot thank you enough! You have NO idea how helpful this is! You guys are absolutely great.”

Here’s one heart-warming story we had to share in which Tech Serv helped a group of students turn frowns into smiles.

In April, Tech Serv received a message from a professor from a university in Michigan regarding an issue with the pGEM Vector System. He was teaching a cell and molecular biology course and his students were unable to generate any colonies. “I have a very disappointed group of seniors on my hands. Please see the photo attached. All those sad faces trying to exude how hard they’ve worked with nothing to show for it. Any insight would be greatly appreciated,” he wrote.

“I understand the frustation of a kit that is not working, the students look so sad!” replied the Tech Serv team. Turns out, the cells may have been past expiration or subjected to repeated freeze thaws that caused the cells to lose competence. Tech Serv sent them a replacement kit with a photo of the team for encouragement.

“We greatly appreciate you replacing what we have and aim to turn those frowns into happy faces before graduation,” the professor replied.

Two weeks later, they got their colonies and wrote back: “It worked very well! We were able to make the most of this and they experienced a very good exercise in troubleshooting. I would say the group would view all that happened as a success. Thank you, we will continue to order from Promega as you’ve always proven to be a very client-friendly company!”

Nothing brings more happiness to the Tech Serv team than your success, so don’t hesitate to contact them with any questions you may have. They’re here to help.

Thanks, Tech Serv!

Fun at the Wisconsin Science Festival

“Is this a real human brain?” I asked. The answer was yes. The liver, lungs, spleen and stomach that were on display were also real—all from donated human bodies. My 3-year-old daughter put on a latex glove and eagerly touched each of the organs, while my 6-year-old son stood back at a distance, wide-eyed. We were at the Discovery Expo on the University of Wisconsin-Madison campus, a free kid-friendly science event featuring dozens of interactive exploration stations. Continue reading “Fun at the Wisconsin Science Festival”

My City Flooded, and There’s More to Come

It seemed like the rain was never going to stop. It started in the morning, and when I left work around 5pm, it was still coming down hard. I took my normal route home through a back country road. As I turned right onto Fitchrona Road, a long line of cars came into view. There’s usually some congestion leading to the stop sign ahead. Except today, something was different. About 20 yards of the road ahead was submerged in water. Continue reading “My City Flooded, and There’s More to Come”

Autophagy: The Poem

Roberta A. Gottlieb, MD, is the Director of Molecular Cardiobiology at Cedar-Sinai, a nonprofit academic healthcare organization. She is interested in the role of autophagy in myocardial ischemia, a kind of heart disease in which blood flow to the heart is blocked. (Studies have shown that autophagy is upregulated during myocardial ischemia, but why this happens is not entirely clear.) Her ultimate goal is to understand and mitigate ischemic injury, with the hope of developing therapeutics for humans.

And—she’s a poet. Continue reading “Autophagy: The Poem”

High-Throughput Drug Screening Using 3D Cell Cultures

For a long time, the drug industry has relied on flat 2D cell cultures grown on a plate to screen for potential drugs. However, 2D models do not accurately reflect the native environment of cells in vivo. 3D cell cultures, on the other hand, better represent the numerous cell-cell and cell-matrix interactions and hypoxic conditions that have a profound effect on the behavior of cells. In a 2018 study published in Oncogene, Kota et al. developed a high-throughput 3D spheroid-based screening assay to identify drug candidates that target RAS proteins.

RAS proteins are GTPases that transmit extracellular signals into cellular signaling pathways, which could activate cell proliferation, differentiation and survival mechanisms. Oncogene mutation in the three human RAS genes (HRAS, NRAS and KRAS) are found in 30% of all cancers, making RAS proteins the most common oncogene. In fact, mutations in KRAS are found in >90% of pancreatic cancers. Despite the prevalence of RAS mutations, targeting RAS proteins with drugs is extremely challenging due to the complex nature of the protein.

The authors in this study wanted to test a new approach using a 3D spheroid-based screening assay to find drugs that target RAS proteins. They first harvested 2D monolayer cultures of pancreatic epithelial tumor cells that express either wild-type KRAS or mutant oncogenic KRAS, and tested their ability to form 3D spheroids. They confirmed spheroid growth using the CellTiter-Glo® 3D Cell Viability Assay with linearity of detection in the range of 1,000–10,000 cells seeded.

The 3D spheroids were then treated with a library of 1,280 known drugs. From the high-throughput screen, they identified one compound with the greatest selective inhibition against oncogenic KRAS. The compound is called Proscillaridin A, a cardiac glycoside that is known for treating congestive heart failure and cardiac arrhythmia. In 3D spheroids, Proscillardin A inhibited oncogenic KRAS at a >90% inhibition rate, with <10% inhibition of wild-type KRAS. In 2D cultures, however, there was no selective inhibition of oncogenic KRAS (inhibition rates for both oncogenic and wild-type KRAS were about 50%). This means that Proscillaridin A would not have been identified as a candidate if the screen was done using only 2D cultures.

Next, the authors wanted to determine how Proscillaridin A impacts tumor cell viability. Could it induce apoptosis in tumor cells? To test this, they used the RealTime-Glo™ Annexin V Apoptosis Assay. This bioluminescent assay is able to detect apoptosis in real time, based on the exposure of phosphatidylserine on the outer leaflet of the cell membrane when apoptosis occurs. Using this assay, they found that Proscillaridin A induced apoptosis at earlier time points and higher rates in 3D spheroids expressing oncogenic KRAS compared with wild-type KRAS. In 2D cultures, there was no difference in the rate of apoptosis.

This study shows that high-throughput screening in 3D spheroids can identify potential drugs that would not have been discovered in a 2D format. This provides hope for finding drugs against difficult target proteins such as RAS.

Reference: Kota S., et al. (2018) A novel three-dimensional high-throughput screening approach identifies inducers of a mutant KRAS selective lethal phenotype. Oncogene. Epub ahead of print.