Ameet Chimote, PhD
Research Associate, University of Cincinnati, Department of Internal Medicine, Division of Nephrology, Cincinnati, OH, USA
Ameet is currently a Research Associate in Dr. Laura Conforti’s laboratory at the University of Cincinnati, where he studies the role of ion channels in T lymphocyte function, primarily in the context of solid tumors. He obtained his degree in Medicine from the University of Nagpur, India and completed his doctoral training where he studied ion transport physiology in disease causation under the guidance of Dr. Peter Lauf at Wright State University in Dayton Ohio. Subsequently, Ameet joined Dr. Conforti’s laboratory as a postdoctoral fellow where he optimized the methodology for isolating tumor infiltrating lymphocytes from head and neck squamous cell tumors and developed various flow cytometry and microscopy based assays to detect ion channel function in blood and biopsy specimens from cancer patients. Ameet then transitioned to the position of Research Associate in Dr. Conforti’s laboratory in 2015 and is currently continuing with his translational research. In addition to his research responsibilities in Dr. Conforti’s laboratory, Ameet is also passionate about mentoring young STEM students in the laboratory and about science communication and outreach.
Learn about Ameet’s research
Title: Defects in potassium channels contribute to reduced immune surveillance in cancers
- Understanding the physiological role of Kv1.3 and KCa3.1 channels in T cell function and learn how their defective function in cancer T cells can lead to decreased immune anti-tumor response.
- Learn about the various experimental methodologies and functional assays to assess T cell function.
Harnessing the immune system has emerged as a powerful therapeutic strategy in oncology. However, the limited ability of cytotoxic CD8+ T cells to infiltrate solid tumors presents a major roadblock to develop effective immunotherapy. Cytotoxic CD8+ T cells, in fact, have to infiltrate solid tumors, attack and kill cancer cells in order to provide an effective antitumor response. CD8+ T cell effector functions depend on Ca2+ influx into the T cell, which is controlled by two potassium (K+) channels: the voltage-dependent Kv1.3 and the Ca2+-activated KCa3.1. Our laboratory studies the contribution of these channels to T cell effector functions in patients with head and neck squamous cell carcinoma (HNSCC). We recently reported a decreased Kv1.3 function accompanied by a decrease in Ca2+ influx in tumor infiltrating lymphocytes (TILs) isolated from HNSCC patients. Furthermore, CD8+ TILs expressing high Kv1.3 levels and showing increased cell proliferation and cytotoxicity preferentially accumulated in the stroma. We also reported a role for K+ channels in regulating CD8+ T cell infiltration in tumors. Various intratumoral factors, especially the nucleoside adenosine limit the accumulation of TILs. We analyzed the migration of CD8+ T cells from HNSCC patients using a 3D chemotaxis assay and observed that adenosine inhibited the chemotaxis of CD8+ T cells from HNSCC patients to a greater degree than CD8+ T cells from healthy individuals. This increased sensitivity of HNSCC CD8+ T cells to adenosine correlated with their inability to infiltrate the tumor and was due to a decrease in KCa3.1 activity. Thus, our data indicate that defects in the K+ channels in T cells limit their effector functions and migration into the tumors, thereby contributing to the reduced anti-tumor immune response. Positive modulators of these channels could improve cancer immune surveillance, thus potentially opening new avenues for cancer immunotherapy.
Watch the Webinar:
Presenter: Ameet A. Chimote, PhD
00:00 – Slide 1
Alexis Corrales: Hello, everyone, and welcome to today’s live broadcast, Defects in Potassium Channels Contribute to Reduced Immune Surveillance in Cancers, presented by Dr. Ameet Chimote, Research Associate, University of Cincinnati Department of Internal Medicine, Division of Nephrology, Cincinnati, Ohio. I am Alexis Corrales of LabRoots and I’ll be your moderator for today’s event.
Today’s educational web seminar is brought to you by LabRoots and sponsored by Thermo Fisher Scientific. For more information on our sponsor, please visit thermofisher.com. Now let’s get started. (00:32)
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This presentation is educational and thus offers continuing education credits. Please click on the Continuing Education Credits tab located at the top right of the presentation window and follow the process to obtain your credits. (01:25)
I’d like to now introduce our presenter, Dr. Ameet Chimote. Ameet is currently a research associate in Dr. Laura Conforti’s laboratory at the University of Cincinnati where he studies the role of ion channels in T-lymphocyte function, primarily in the context of solid tumors. He obtained his degree in Medicine from the University of Nagpur, India, and completed his doctoral training where he studied ion transport physiology in disease causation under the guidance of Dr. Peter Lauf at Wright State University in Dayton, Ohio. (01:55) Subsequently, Ameet joined Dr. Conforti’s laboratory as a postdoctoral fellow, where he optimized the methodology for isolating tumor-infiltrating lymphocytes from head and neck squamous cell tumors and developed various flow cytometry- and microscopy-based assays to detect ion channel function in blood and biopsy specimens from cancer patients. Ameet then transitioned to the position of research associate in Dr. Conforti’s laboratory in 2015 and is currently continuing with his translational research. (02:27) In addition to his research responsibilities in Dr. Conforti’s laboratory, Ameet is also passionate about mentoring young STEM students in the laboratory and about science communication and outreach.
For a complete biography on our speaker, please visit the Biography tab at the top of your screen.
Dr. Chimote, you may now begin your presentation. (02:49)
Ameet Chimote: Thank you for the great introduction and hello, everybody. My name is Ameet Chimote and I am a research associate in the lab of Dr. Laura Conforti at the University of Cincinnati. On behalf of myself and my mentor, Dr. Conforti, I am very excited to get this opportunity to be in front of all of you and present our research to you. (03:09)
First of all, I want to start by thanking Gibco for giving me this wonderful opportunity and this platform to present our research. The Conforti lab studies the contribution of ion channels in how T cells function, especially in cancers. In this webinar, I want to shine a spotlight on how potassium ion channels in T lymphocytes contribute to immune responses in cancers.
(03:41) – Slide 2
I would like to begin this talk by acknowledging all of our wonderful contributors and collaborators who have contributed to these studies, particular Dr. Trisha Wise-Draper in the Division of Hematology-Oncology at the University of Cincinnati, who has given us the patient samples and also all the clinical data related to the patient samples, and has been vital in progressing these studies. And this is just a picture of our research building on the beautiful, beautiful University of Cincinnati campus.
(04:10) – Slide 3
So first of all, I want to start by talking a little bit about the immune system and its role in cancer causation as we all know that the immune system is extremely important in causing cancer. In order to develop new immunotherapies that harness the individual’s own immune system to fight cancer, it has been very paradigm-shifting in the field of discoveries that relate to fighting cancers in the past few years. (04:40)
In order to develop these immunotherapies, it is very vital that we understand how the immune system works, and this has been made possible mainly because of all the hard work done by the basic scientists and the translational scientists and the clinical scientists in the field. (04:58)
Our laboratory mainly studies the T cell function in the tumor microenvironment in head and neck cancers.
(05:05) – Slide 4
Let me start by talking and introducing the head and neck cancers. So head and neck cancers are the sixth most common malignancies and they are associated with significant mortality rate. Head and neck cancers are characterized by the presence of solid tumors in the oral cavity, the nasopharynx, oropharynx, hypopharynx and the larynx. As with most of the cancer types, the common modalities of treatment for head and neck cancers are surgery, radiation therapy and chemotherapy. (05:42)
However, because of the anatomical location of these tumors, the treatment modalities of surgery and chemotherapy are associated with a lot of impairment of basic life-sustaining functions in these patients such as swallowing or speech, and because of the cancer and also because of the treatment modalities, this type of cancer has a 50% mortality rate. (06:09)
The majority of the head and neck cancers are squamous cell carcinomas, HNSCCs.
Currently, there are immunotherapies, mostly immune checkpoint inhibitors, which have been approved in the treatment of immunotherapy and they have indeed shown promise in the treatment of these head and neck cancers. However, they are effective only in a small population of the head and neck cancer patients and therefore there is an ongoing focus of research on developing new and improved immunotherapies which can work and which can be effective for a wide variety of patients. To understand the challenges that are faced by the immune system to produce the antitumor immune response, let me start by talking a little bit about the composition of the tumor microenvironment.
(06:59) – Slide 5
What I’m showing here is a very, very simplified scheme of the tumor microenvironment. It is illustrative and it highlights the key components of the tumor microenvironment but in no way this is an exhaustive list of the components of the tumor microenvironment. (07:15)
So for the sake of this presentation, the tumor microenvironment is composed of cellular components and noncellular components, both of which are in interplay with each other and produce the classic hallmarks of cancer. These cellular and noncellular components of the tumor microenvironment, they allow the tumor cells to proliferate, to grow, to metastasize and, most importantly, they allow the tumor cells to evade the immune system. (07:51)
It is also worth noting that the tumor microenvironment is very dynamic; that is, the components and the cells, they keep changing dramatically, and this can change how the tumor progresses.
For the sake of this presentation, I am going to mainly focus on the cellular component and mainly the immune cells and in the second part of this talk, I am going to talk about how the noncellular component, which is adenosine, highlighted in red here, will affect the response of the immune cells and potentially inhibit the immune cells. (08:29)
Let me first start by talking about the immune components of the tumor microenvironment.
(08:35) – Slide 6
So the tumors are infiltrated by cells of both the innate as well as the acquired immunity such as the myeloid-derived suppressive cells, macrophages, dendritic cells, mast cells, eosinophils, neutrophils, NK cells as well as the lymphocytes.
The tumor-infiltrating lymphocytes are both CD4+ as well as the CD8+ T lymphocytes. They both have a very distinct function. For the sake of this presentation, I am going to talk mainly about the tumor-infiltrating lymphocytes which express the CD8 on their membrane, which are the cytotoxic or the CTLs. (09:20)
Why are they important? The cytotoxic CD8+ T cells bind directly to the tumor cells and they mediate tumor cell killing by cytokine release and cytotoxic granule release. They are usually the first line of defense against a tumor by our body.
To perform these cytotoxic functions, it is very important that these CD8+ T cells first of all, they migrate towards the tumor microenvironment, then they infiltrate the tumor microenvironment and third, they identify and then kill the tumor cells by release of cytotoxic granules. (10:00)
The cytotoxic functions of the T lymphocytes depend on calcium signaling. Calcium signaling, in turn, is mediated by the various ion channels which are present in the T lymphocytes.
(10:14) – Slide 7
In this slide, I am going to talk in detail about the role played by ion channels in the calcium signaling in the T lymphocyte and how it is very vital to produce the desired cytotoxic effect. So as you can see here in this figure on the left, the calcium signaling in the T cells is regulated mainly by two potassium channels, one calcium channel and one channel (TRPM7), which can allow both the calcium and magnesium to come in the cells. (10:46)
The two important types of potassium channels which are involved in the calcium signaling of the T lymphocyte are the voltage-gated Kv1.3 channels and the calcium-dependent KCa3.1 channels.
The calcium channel that you can see here is called as the CRAC channel, which is the calcium release activated channel, and this comprises of two main subunits. There is the ORAI1 subunit which is present in the cell membrane and there is the STIM1 subunit which is present intracellularly near the endoplasmic reticulum. (11:25)
To begin with, the intracellular calcium levels are very low. They are in the nanomolar range. So let me now recount what are the cellular events that happen when a T lymphocyte is activated and how the calcium signaling takes place.
So we first start with the T cell receptor. When the T cell receptor is engaged, the T cell receptor is activated and it will start a cascade of signaling events which begins by release of inositol triphosphate. This inositol triphosphate, it will cause release of calcium from the endoplasmic reticulum. (12:00)
It is very important to note that this amount of calcium that is released through the endoplasmic reticulum is very, very little. It’s only in the nanomolar range and by itself, this calcium cannot trigger any of the downstream signaling events which can cause cytokine production or any kind of effector T cell activity.
However, what this release of calcium from the endoplasmic reticulum stores does is that it will activate the STIM1 subunit and this oligomerizes the STIM1 subunit and makes it to translocate to the cell membrane where it binds with the ORAI1 subunit, which allows for the calcium to enter through the CRAC channel. As I had said before, the CRAC channel is formed when the ORAI1 and the STIM1 subunits interact together. (12:50)
So now through this CRAC channel, the calcium comes in and the amount of calcium inside the cell increases from the nanomolar range to the micromolar range. To sustain this calcium influx, which is the influx of a large amount of positively charged ions into the cell, the cell has to balance somehow by efflux of positively charged ions from the cell. This efflux is achieved through the potassium channels, which allow for an efflux of the potassium ions from the cell. (13:20)
The first potassium channel to activate is the voltage-gated Kv1.3 channel and this prevents the cell from depolarization, and this will allow for the calcium entry through the CRAC channel to be sustained. Now, as more and more calcium accumulates in the cell, this calcium will activate the second potassium channel, the calcium-activated KCa3.1 channel, and this causes further efflux of potassium through the cell, which keeps the membrane hyperpolarized and more and more calcium keeps on coming in the cell through the CRAC channel. (13:56)
Now that the calcium levels in the cell increase, it allows for the activation and nuclear translocation of the NF-AT, which in turn increases the transcriptional activity of several transcriptional factors and also it causes for increased cytokine production and cell proliferation.
So just to highlight the functions of the Kv1.3 and KCa3.1 channels, we have the table on the right. So both the Kv1.3 as well as the KCa3.1 channels are responsible for the proliferation of the T cells. They are responsible for cytokine production and they are also responsible for cytotoxicity. Previous research from our lab has also shown that in human T cells, the KCa3.1 channels are very important and involved in the migration of these T cells. (14:49)
As vital as these channels are to T cell function, we do not know what is the status of these potassium channels in the immune cells that have infiltrated the tumor microenvironment, and this was the purpose.
So we first undertook studies to investigate what is the function of these ion channels in T lymphocytes from head and neck cancers. So first let me talk about what is the function of Kv1.3 channels in the tumor-infiltrating lymphocytes and in the cancer T cells.
(15:18) – Slide 8
So this is a summary table of the patient profile of the head and neck cancer patients that we received from the clinic at the University of Cincinnati Medical Center. The inclusion criteria that we used for these patients were that these patients had to have a positive diagnosis of head and neck cancer confirmed by histopathology and at the time of blood and sample collection, these patients should not have received any chemotherapy or radiation therapy. As always, we obtained informed consent from all of the patients.
(15:54) – Slide 9
So in the next slide, in this slide, I’m presenting an outline of how we processed the samples to study the Kv1.3 channel function as well as the Kv1.4 channel expression in the tumor-infiltrating lymphocytes.
So first from the head and neck cancer patients, we obtained the blood. From the blood, we isolated peripheral blood T cells. Then, from these patients, we also obtained the surgically resected tumor. The surgically resected tumor was used for two different experiments.
Part of the tumor, we isolated tumor-infiltrating lymphocytes, and in the next slide I’m going to talk in detail about how we isolated the tumor-infiltrating lymphocytes from the solid tumor. (16:37)
In the peripheral blood T cells as well as the tumor-infiltrating lymphocytes, we performed functional studies. The other half of the tumor we froze and prepared frozen sections from that, and from these frozen sections, we did immunohistochemical staining and performed confocal microscopy to determine the extent of T cell infiltration, the expression of Kv1.3 channels as well as to assess cytotoxicity and cell proliferation. (17:06)
For performing the functional studies, we used peripheral blood T cells from healthy donors as controls.
(17:14) – Slide 10
So in this slide, I am just talking about, in brief, about how we isolated the tumor-infiltrating lymphocytes for the functional studies and in the manuscript that we have here, in the bottom of the page, you will find much detailed description of how we did it. (17:33)
So in brief, we obtained the tumor sample from the OR and we processed these samples immediately or, at the most, within two hours of their surgical resection. First, we rinsed these tumors with phosphate-buffered saline and we chopped them into small 2 mm pieces.
Next, these fragments we put in RPMI medium and they were mechanically dissociated. After mechanical dissociation, we filtered them through a 100 micron filter and we obtained a single-cell suspension. From the single cell suspension, we layered it over a Ficoll density gradient and we obtained mononuclear tumor-infiltrating lymphocytes. (18:19)
And then from these mononuclear cells, we further did a purification of CD3+ tumor-infiltrating lymphocytes by using a commercially available negative selection kit for cell isolation.
(18:36) – Slide 11
So when we first isolated the cells, the first experiment that we did was we wanted to characterize the purity and the phenotype of the tumor-infiltrating lymphocytes. So this was done mainly by antibody staining and flow cytometry. As you can see in the first plot, we see that the tumor-infiltrating lymphocytes were over 99% viable and they were predominantly CD3+. (19:02)
Out of the CD3+ tumor-infiltrating lymphocytes, we saw that 56% of these were CD4+ whereas 35% were CD8+. Then we looked at the CD4+ tumor-infiltrating lymphocytes and we saw that 82% of these were effector memory T cells and 25% of the CD4+ tumor-infiltrating T cells were regulatory T cells, much higher than the amount present in noncancer patients, but this is commensurate with the number that’s reported in the literature. (19:43)
Now that we found the functional phenotype of the tumor-infiltrating lymphocytes, we wanted to look at the expression levels and the function of Kv1.3 channel as the function of these ion channels in the tumor-infiltrating lymphocytes is not known.
(20:01) – Slide 12
So the first experiment that we did to study the Kv1.3 channel activity was to study Kv1.3 currents using electrophysiology. In electrophysiology, we measured the currents using whole-cell patch clamp technique. (20:19)
So as you can see in the graph on the top left, we see that the Kv1.3 currents are significantly reduced in tumor-infiltrating lymphocytes as compared to the peripheral blood T cells which were isolated from the same donor. (20:37)
These are the summary of four individual patients as shown in the graph on the right-hand side, and here we can see that again, summary of four patients, the currents in tumor-infiltrating lymphocytes are significantly lower as compared to peripheral blood T cells from the same individual. However, the Kv1.3 channel activity which is present in the peripheral blood T cells from cancer patients is not significantly different than the Kv1.3 channel activity in peripheral blood T cells from healthy donors. (21:11)
So Kv1.3 channels, as I explained before, are very important in regulating the calcium signaling and the calcium influx through the CRAC channel. So since we saw that the Kv1.3 channel activity is reduced, we wanted to see downstream whether the calcium influx through the CRAC channels is affected or not because of the decreased function of the Kv1.3 channels. (21:38)
So as you can see in the bottom figure, we measured the calcium influx in these T cells and the tumor-infiltrating lymphocytes using Indo-1 ratiometric values for measuring calcium and then performed flow cytometry. We measured the calcium influx in these cells using the calcium add-back method. Let me talk just a little bit about how we did it, and there are more details about this in our publication; the link is on the right corner. (22:05)
So initially, we loaded the cells with this Indo-1—the peripheral blood T cells as well as the tumor-infiltrating lymphocytes from the same individual, we loaded those with the Indo-1 ratiometric dye. After that, the cells were kept in a calcium-free solution. (22:22)
Now, when the cells were recorded in the calcium-free solution, we see that the Indo-1 fluorescence is low, which is the baseline. Then, we added thapsigargin, which is a SERCA pump inhibitor, which is at the position of the arrow on the graph. Thapsigargin, by inhibiting the SERCA pump, it will cause release of calcium from the intracellular stores, which should trigger the signaling cascade which I mentioned in the earlier slide and cause an influx of calcium through the CRAC channels. However, the cells are still in a zero millimolar calcium outside so we do not see any significant rise in the Indo-1 ratio. (23:03)
Once this recording progresses, we change the extracellular solution and add 2 millimolar calcium, which is a very high amount of calcium, extracellularly. At this point, because of the addition of the thapsigargin, the CRAC channels are already open and there is a massive influx of the calcium. However, interestingly, as we can see in this graph, the calcium intake is decreased in the tumor-infiltrating lymphocytes as compared to the peripheral blood T cells, which is also summarized in the fold change in the calcium on the right-hand side, that we see that in the tumor-infiltrating lymphocytes the calcium influx is decreased as compared to peripheral blood T cells from the same patient, and which is not different in turn from the calcium response that we see in healthy donors. (23:54)
So now that we determined that, okay, the Kv1.3 channel function is reduced, we wanted to see what is the expression profile of Kv1.3 channels in the tumor-infiltrating lymphocytes in solid tumors.
(24:07) – Slide 13
So we looked at the infiltration of these T cells using immunofluorescence techniques. As I said earlier, we obtained frozen sections from the tumor samples and we stained them with pan-cytokeratin, which is PCK, which is a marker for the tumor epithelium. Then we stained them for CD8, nuclei were stained with DAPI, and then the Kv1.3 antibody—Kv1.3 channel—was stained with the antibody. (24:35)
So first of all, we localized where the CD8 infiltration is actually taking place and for that, we used pan-cytokeratin staining as a determinant.
So the CD8 cells which were present in the area within pan-cytokeratin staining were considered to be intratumoral and are designated in the graph below as iTILs. All the other CD8 cells were considered to be stromal tumor-infiltrating lymphocytes and are designated as sTILs. (25:07)
As we observe in the graph which is in the middle, we see that the majority of the CD8+ T cells were stromal in nature.
So now, in these intratumoral and stromal cells, we wanted to look at the expression levels of Kv1.3 channels. In order to determine that, we looked at the CD8 cells and within all of the CD8 cells, we measured the mean fluorescence intensity of the Kv1.3 signal. And then, as you can see in the bottom left graph, we ranked all the Kv1.3 intensities within the CD8+ T cells from low to high, and then we took the median fluorescence intensity of Kv1.3. (25:48)
All the cells that had Kv1.3 channel fluorescence intensities above this median were designated as Kv1.3 High, and all of the cells which had Kv1.3 fluorescence below this median value were designated as Kv1.3 Low.
We found out through this analysis, and which is summarized on the graph to the right-hand side on the lower column, in the lower row, that the percentage of Kv1.3 High CD8+ T cells were mainly stromal as compared to intratumoral. Thus, these data show us that CD8+ T cells not only fail to penetrate into the tumor—that is the intratumoral cells—not only CD8+ cells are not only less in amount but they also possess a lower level of Kv1.3 expression.
(26:56) – Slide 14
So as I said earlier, Kv1.3 channels control the effector functions of T cells.
So we wanted to see whether this downregulation of Kv1.3 channel expression in the CD8+ tumor-infiltrating lymphocytes was associated with any kind of reduction in the effector function. (27:17)
For this purpose, we assessed the following two effector functions of the cytotoxic T cells. We first look at the cell proliferation and then we also looked at the granzyme B production, which are both very important for the CD8+ tumor-infiltrating lymphocytes to attack and kill the tumor cells. (27:37)
So we stained the tumor cells with pan-cytokeratin, which is a marker for the tumor epithelium, then we stained them with—stained all of the CD8 cells with the CD8 antibody. We stained granzyme B with the antibody. As a marker of proliferation, the sections were stained with a Ki-67 antibody while the channel was stained with a specific antibody against the Kv1.3 channels. (28:04)
So we did these experiments in nine donors and again, as I said earlier, using the median fluorescence, using the median fluorescence calculation, we calculated which of the CD8+ cells in these sections from nine individuals were high in Ki-67 expression and also high in granzyme B production. (28:30)
So we assumed that the section—the cells which are high in Ki-67 are the proliferating cells. And then we looked at the percent high granzyme B-producing cells. In both of these cell populations, we looked at the CD8 cells which were also Kv1.3 High and Kv1.3 Low.
Interestingly, we observed that the percentage of proliferating cells, again which were high in Ki-67 and granzyme B, were higher in CD8+ TILs, which expressed higher amounts of Kv1.3. Thus, to conclude or just to sum up this slide, we see that higher the Kv1.3 expression in CD8+ cells is associated with a more proliferating state, and these—but as we saw in the earlier slide, these cells, which are highly proliferating and producing more granzyme B, are located mainly in the stroma and not inside the tumor.
(29:40) – Slide 15
So just to summarize, the role of Kv1.3 in head and neck cancer tumor-infiltrating lymphocytes, the head and neck solid tumors are infiltrated with tumor-infiltrating lymphocytes and they are preferentially in the tumor stroma. These tumor-infiltrating lymphocytes, they show a loss of Kv1.3 channel function which ultimately limits the calcium influx. The Kv1.3 expression is downregulated in the intratumoral tumor-infiltrating lymphocytes. Lower Kv1.3 expression is associated with reduced proliferation and granzyme B production. (30:20)
So to sum up, this is the way that Kv1.3 channels and the Kv1.3 channel expression in the tumor-infiltrating lymphocytes, they mark the functionally competent cytotoxic TILs in head and neck cancer. However, even if the Kv1.3 expression is high in these cells, these cells somehow have to migrate towards the tumor and infiltrate the tumor to produce their desired function and this is what I want to talk about in the second half of my talk.
(30:51) – Slide 16
So just to sum up, we looked at the cellular components, which are the immune cells, in the tumor microenvironment, but the migration of these immune cells within the tumor microenvironment is influenced by a lot of extracellular components, mainly adenosine. So the question that we asked is: how do these extracellular components of the tumor microenvironment limit the infiltration of the tumor-infiltrating lymphocytes into the tumor and also impair their function. And I want to mainly talk about adenosine, and here’s why.
(31:31) – Slide 17
So the tumor microenvironment is very rich in adenosine. Adenosine is a purine nucleoside which is secreted in very large amounts by tissue due to inflammation and, in the case of tumors, because of tumor hypoxia. The amount of adenosine in the tumors, solid tumors especially, is increased almost one hundredfold, and this excessive accumulation of adenosine contributes to the failure of effector T cells to eliminate cancer cells. (32:00)
It is very well-known that the elevated adenosine levels in solid tumors is associated with tumor progression, also associated with enhanced tumor metastasis as well as resistance to therapy and ultimately a very poor prognosis. (32:17)
Recent studies have also highlighted that inhibiting the signaling pathway of adenosine improves T cell function and it will also reduce the tumor burden.
(32:30) – Slide 18
So what is the effect of adenosine on T lymphocytes? So adenosine inhibits the T lymphocytes and acts on the T lymphocytes via the A2A adenosine receptor, which is a G protein-coupled receptor. And adenosine is known to inhibit activation of T cells, proliferation of T cells, cytotoxicity of T cells as well as motility of T cells, which are in fact all of the effector functions of T cells. (32:58)
It is very critical that the T cells migrate towards the solid tumors which are rich in adenosine to mount an effective immune response. However, this is inhibited.
We have published previous studies in which we have shown that adenosine inhibits the function of KCa3.1 channels in human T cells, and this inhibition takes place via A2A adenosine receptor. (33:29)
The scheme on the right is basically a cartoon of a migrating T cell, and the T cell is migrating towards a stimulus via chemokine or some kind of a factor, in the direction of the red arrow that you see here. So a T cell migrates in an amoeboid fashion and while migrating, the part of the T cell that migrates towards the chemical stimulus is called as the leading edge, whereas the rear end of the T cell is called as the uropod. The ion channels, interestingly, get polarized in a migrating T cell. (34:05)
About the potassium channels, the Kv1.3 channels are present in the leading edge, whereas the calcium-dependent KCa3.1 channels are present in the uropod, and previous studies from our lab have shown that these KCa3.1 channels which are present in the uropod are involved in the migration of the T cells. (34:28)
So the main question that we ask here is that how are these ion channels affected in the presence of adenosine so as to affect their movement in a three-dimensional tumor microenvironment kind of milieu. So this is what we wanted to look at.
(34:47) – Slide 19
And this is how we did it. So this is the summary of the various patients that we got from the clinic again, and we had the same inclusion criteria for this study as before, that the patients should not have—the patients should not have received any chemotherapy or radiation therapy at the time of sample collection, and a diagnosis of head and neck squamous cell cancer should have been confirmed by histopathology. (35:13)
So from all of these patients, of course after obtaining informed consent, we obtained blood samples and from the peripheral blood of these patients, we isolated the peripheral blood T cells. These peripheral blood T cells were activated for 72-96 hours using a plate-bound anti-CD3/anti-CD28 antibodies, and this will cause an activation, a T cell receptor-mediated activation basically. (35:41)
And these activated CD8+ T cells were used for the further studies that I’m going to talk about, and peripheral blood T cells which were isolated and activated the same way from healthy donors were used as controls.
So now, what we did was we took these activated CD8+ T cells from cancer patients and healthy donors, and we looked at the ability of these peripheral blood T cells to migrate in a three-dimensional environment using a 3D chemotaxis assay.
(36:12) – Slide 20
So I’m going to spend a little bit of time on explaining how we did this 3D chemotaxis assay and I am just going to go step by step.
So just to give a little bit of background, the tumor microenvironment is characterized by the presence of tumor cells as well as immune cells which are present in a fibrous extracellular matrix. In this matrix, these immune cells and the tumor cells are exposed to a wide variety of extracellular factors which are present in the tumor microenvironment such as chemokines, cytokines, adenosine which I had explained before. (36:51)
So now we wanted to mimic this environment in vitro, and in order to do this in vitro, we used the assay called as μ-Slide 3D Chemotaxis assay from the company ibidi, and there is a link on the bottom of this slide which will tell, which can tell you in more detail about how this assay works and how it is done, but let me just talk about it just a little bit, okay. (37:17)
So this μ-Slide 3D Chemotaxis chamber, it allows us to study the migration of CD8+ T cells which are activated in a stable 3D collagenous matrix, and this allows us to expose the cells to a chemokine as well as to adenosine or some other compounds, and we can look at the desired study. Here’s how we did it. (37:40)
The cells are suspended in a collagenous matrix, and this collagenous matrix is shown in the yellow channel in the center. So imagine in this yellow channel, there are the cells which are in a collagen gel, kind of like a tube, like a three-dimensional. Now they are exposed to two different kind of chambers. In the right chamber, which is in blue color, we add just plain media. In the chamber on the left, we add the media as well as a chemokine or, in some of the cases, we add chemokine along with adenosine or some kind of analogs of adenosine. (38:19)
So just to simplify this, on the right-hand side well, there is no chemokine. On the left-hand side well, there is a large amount of chemokine and the cells are in the middle. So now the chemokine is causing a gradient and the cells are exposed to this gradient. Ideally, the cells should start migrating towards the chemokine and this migration we detect and measure using time-lapse microscopy. Then, these cells are tracked using a tracking software and we can create a path of each and every cell. (38:55)
And we have shown here a representative chemotaxis experiment on the far right. So as you can see here, we have a chemokine gradient, CXCL12 gradient from top to bottom, shown with the green triangle. And if we track the individual tracks of 20 cells which begin from a common point of origin [0,0], you can see that visually, that a lot of these cells are going in the direction of the y-axis where the chemokine gradient is present. (39:25)
To quantitate this chemotaxis, we used a measuring parameter called as center of mass. So what the center of mass does is that it takes the coordinates of the endpoints of all of the migrating cells which have started from a common point of origin which is the [0,0] coordinate on this axis that you can see here in this graph. (39:46)
Then, from these endpoints, it will calculate a spatial endpoint which will be—one point which will represent the net direction where all of these cells have migrated. Now, since our chemokine gradient is around the y-axis, if this net point, the Y center of mass, which is shown by this blue plus sign, is more, a higher positive number on the y-axis, it would mean that most of our cells have gone in the direction of the chemokine gradient, which indicates a positive chemotaxis. (40:17)
However, if this number is very close to the origin or a negative number on the y-axis, it would mean that the cells, the net direction of the cells is not going towards the high concentration of the chemokine, thus the cells are not undergoing chemotaxis and it would signify a negative chemotaxis. This is how we have interpreted the data.
(40:42) – Slide 21
So here’s what we did. We isolated and activated CD8+ peripheral blood T cells from healthy donors as well as head and neck cancer patients, and we subjected them to a gradient of the chemokine CXCL12 as well as a gradient of CXCL12 and adenosine, and we measured their chemotaxis. (41:03)
And what we are showing in the plots in the middle are the Y center of masses in both these conditions. So let me take you through this slide.
So first, what we see is that in the healthy donors, they are undergoing chemotaxis towards the chemokine gradient as seen by the high amount of the Y center of mass. However, we see only a 26% decrease in the Y center of mass when an adenosine is also added along with the chemokine gradient. So in the presence of adenosine, the chemotaxis of healthy donors is not significantly affected. (41:41)
On contrary, the chemotaxis in head and neck cancer patients in the presence of chemokine only is comparable in values to the healthy donor values. However, when we add adenosine along with the chemokine gradient, the cells do not migrate towards the chemokine and there is an 80% inhibition in the chemotaxis that you can see here. Thus, this is a very significant inhibition of chemotaxis only in the peripheral blood of head and neck cancer patients. (42:18)
And so we wanted to see why is it so? Like we wanted to look at the mechanism and we wanted to look at the implications of this. What does it mean? So we wanted to first test whether the accumulation of adenosine in the solid tumors would indeed inhibit the migration of the CD8+ T cells into the tumor.
(42:39) – Slide 22
So here’s what we did. We obtained biopsy sections from all of the patients whose chemotaxis we had studied, and we stained these sections using immunohistochemistry for CD8 and also we stained them for CD73. So CD73 is an enzyme which is required for the production of adenosine and it is very commonly used as a marker for adenosine. So if any tumor is high in CD73, it is assumed that the tumor is high in adenosine production. (43:13)
So first of all, we looked at the CD8 expression of these tumors where we had measured the chemotaxis and we classified these tumors as CD8 High or CD8 Low based on the infiltration of CD8 cells per mm2. And as you can see, all of these tumors presented with a very variable amount of CD8 infiltration. (43:36)
Next, we classified the adenosine levels in these tumors based on CD73 expression using CD73 High and CD73 Low. We then looked at the CD73 High tumors and in these CD73 High tumors, we plotted what you can see in the extreme right-hand side graph. We plotted the infiltration of CD8 as a function of the inhibition of chemotaxis. (44:04)
And interestingly, what we saw, that in these tumors which express very high amounts of adenosine, the amount of inhibition of chemotaxis was highest in those tumors which showed a very low infiltration of CD8 cells in the tumor. So this was a very significant finding and then we wanted to look whether this inhibition of chemotaxis of CD8 cells to infiltrate the tumor is a process which is mediated by the A2A adenosine receptor.
(44:36) – Slide 23
So it is very well-known that the adenosine receptor which is involved in T cells is the A2A adenosine receptor and it is a G-protein-mediated—G-protein-coupled receptor. And we conducted experiments to see whether the effect of adenosine on the head and neck cancer peripheral blood T cells which inhibits the chemotaxis in these cells is via the A2A receptor or not. So this is the first experiment we did. (45:08)
So instead of, so we had peripheral blood T cells which were activated from head and neck cancer patients and we exposed them to a gradient of CXCL12 and you can see here that they are undergoing chemotaxis towards the chemokine gradient. (45:23)
Next, we exposed them to a gradient—these cells to a gradient—of the chemokine as well as CGS21680, which is an agonist of A2A receptor, which will stimulate the receptor directly. And here we see that as soon as the A2A adenosine receptor is directly stimulated, the chemotactic response is inhibited, comparable to what would happen if the cells are exposed to a gradient of CXCL12 as well as adenosine. (45:56)
So this gives us the first hint into telling that if you stimulate the A2A receptor directly, the chemotaxis is inhibited.
To confirm, what we did was we blocked the A2A adenosine receptor using the A2A receptor antagonist SCH58261. So in these cells, where the A2A receptor was blocked, we see that there is not an inhibition of the chemotaxis in the presence of adenosine as comparable to if it would have just been adenosine as compared to the A2A antagonist. (46:40)
To simplify—sorry, I fumbled here a little bit—when the A2A receptor is blocked, in the presence of adenosine, the chemotaxis is not inhibited as much.
So the results of both these experiments, they show that the inhibition of chemotaxis in the presence of adenosine is mediated mainly by the A2A receptor.
(47:04) – Slide 24
So then we did experiments to see whether, why is this inhibition happening? a) Is this inhibition happening because of any change in the expression of the A2A adenosine receptor itself, or is it because of some kind of inhibition that is happening downstream of the A2A receptor? (47:22)
So first we looked at the A2A receptor expression using qPCR and flow cytometry, and we saw that by qPCR, there was no change in the A2A receptor expression in healthy donors as well as the head and neck cancer patients. And we confirmed this using flow cytometry, that there was absolutely no change in the A2A adenosine receptor expression. (47:46)
So downstream of the A2A receptor, the adenosine signaling pathway should induce an increase in cyclic AMP, and we see that in the healthy donor as well as in the HNSCC patients there is no change in the cyclic AMP expression, as well as there is no change in the downstream protein kinase A activity also. (48:10)
Now, with the activation of PKA, the downstream event that happens in the adenosine signaling pathway is that it will inhibit the KCa3.1 channel. So this is what we wanted to look at next.
So the question that we asked is the inhibition of chemotaxis that we see in the presence of adenosine, is it because of inhibition in the KCa3.1 channel in the head and neck cancer peripheral blood T cells?
(48:37) – Slide 25
So first we looked at the activity of the KCa3.1 channel using electrophysiology as we had seen before for the Kv1.3 channel. So we did electrophysiology here for the KCa3.1 channels using whole-cell patch clamp. (48:54)
And we see here that the KCa3.1 currents are significantly reduced in head and neck cancer patients as compared to the peripheral blood T cells in healthy patients. However, it is very interesting to see that this reduction in the KCa3.1 channel function is not associated with any difference in the KCa3.1 channel expression which we measured using flow cytometry. So now there is a functional defect in the KCa3.1 channels but the expression is almost equal. So we wanted to look at the functional—so we wanted to do more functional studies to confirm this.
(49:36) – Slide 26
For this, we did experiments with an activator of the KCa3.1 channel which is 1-EBIO. So the question that we asked here was if we are seeing an inhibition of the 3D chemotaxis towards a chemokine and adenosine gradient in the head and neck cancer patients and if we assume that this defect is because of a decrease in the KCa3.1 channel activity. So if we stimulate the KCa3.1 channel function, would this defect in the chemotaxis be corrected? So this is what we wanted to look at. (50:12)
So first of all, we wanted to test the compound 1-EBIO and as you can see here, in both healthy donors as well as in the head and neck cancer peripheral blood T cells, addition of 1-EBIO increased the KCa3.1 currents.
Next, what we did was we looked at the chemotaxis of the cells which were exposed—which were pre-incubated in 1-EBIO where the KCa3.1 channels were activated, and we exposed them to a gradient of CXCL12 as well as adenosine. (50:45)
So in this graph, as you see here, there is chemotaxis, positive chemotaxis, that is happening when the cells are exposed to a chemokine-only gradient, and the chemotaxis is decreased significantly when the cells are exposed to a gradient of chemokine, CXCL12, as well as with adenosine.
However, when the KCa3.1 channels are activated in these patients with 1-EBIO and then these cells are exposed to a gradient of chemokine and adenosine, there is no inhibition of the chemotactic response. Thus, it rescues the lack of chemotaxis in the head and neck cancer patients when exposed to an adenosine gradient.
(51:31) – Slide 27
So to summarize the effect of adenosine on KCa3.1 in head and neck cancer CD8 T cells, we see that the CD8+ T cells from cancer patients show a reduced chemotaxis in the presence of adenosine. This process is mediated by A2A adenosine receptor, and this could contribute to the inability of these T cells to penetrate the adenosine-rich solid tumors, and this could arise because of the reduced KCa3.1 activity. (52:05)
Another significant thing that we observed was activation of these KCa3.1 channels in head and neck cancer patients restored the ability of these cells to chemotax towards an adenosine gradient, thus restoring their chemotactic ability.
(52:25) – Slide 28
So to summarize, these are the two main defects—these are the defects that we see in the potassium channels in CD8+ cells in head and neck cancers. First of all, the defect in Kv1.3 channels in head and neck cancer results in a reduced calcium influx as well as decreased cytotoxicity. And nextly, the defect in KCa3.1 channels results in decreased chemotaxis in an adenosine-rich environment and this can possibly lead to decreased T cell infiltration in solid tumors. (53:00)
So why is it important? This is very important because if we use positive modulators of the KCa3.1 channels or if we use modulators of Kv1.3 channels or inhibitors of the A2A adenosine receptor, then we can increase the ability of the CD8 cytotoxic T cells to produce their effector function against the tumor and also it will improve their ability to penetrate the solid tumor to produce the desired cytotoxic effect. And thus, it would be a very important step up in the research for finding effective solutions for solid tumor immunotherapy.
(53:45) – Slide 29
With this, I want to acknowledge our lab members that you see here in the picture, and also I want to acknowledge the different clinical coordinators as well as the Tumor Bank with the University of Cincinnati Cancer Institute, who have provided us with the samples. Also, I want to thank all the Cores where we could do our experiments. The funding was obtained from NIH R01 grant. (54:11)
I want to thank Dr. Conforti for her mentorship and her guidance in completing this project and last but not the least, I am very grateful for all the patients and healthy donors who have participated in this study.
And again, I want to thank Gibco for giving me this opportunity for presenting our research in front of you and with that, I am open to taking any questions from you. Thank you.
Question: Thank you, Dr. Chimote, for your informative presentation.
Ameet Chimote: Thank you. (54:42)
Question: We will now start the live Q&A portion of the webinar. If you have a question you’d like to ask, please do so now. Just click on the Answer a Question box located on the far left of your screen. We’ll answer as many questions as we have time for. So let’s get started.
Our first question is: how did you quantify the staining in the slide before, and did you count every cell stained?
(55:11) – Slide 14
Ameet Chimote: Yes, actually, for example, we have the staining in this particular slide. So what we wanted to look at was we wanted to look at the expression of the granzyme B as well as the Ki-67 and Kv1.3 in the CD8 cells which were infiltrated in the tumor section. So what we did was from each slide, from each patient, we stained it and we did ten, a total of ten fields per section and we quantitated this in all of the CD8 cells. So this was done in all the CD8 cells and the MFI was measured within the CD8s. And there is more information for the procedure in our cancer research paper which is cited on the bottom of this slide. Thank you. (55:49)
Question: Now, Dr. Chimote, why did you study only CD8 T cells and not CD4 T cells?
Ameet Chimote: Well, for the first half of the paper which we had published in which we had the staining and the tumor-infiltrating lymphocytes, we have studied the CD4+ cells also. But mainly what we were interested in are the CD8+ cells because they are the cytotoxic T lymphocytes. They are the first line of defense of the body against the tumor cells, hence their function is very, very important. (56:22)
Also, the CD4+ cells are of dual function, so you have the CD4+ helper T cells and you also have the regulatory T cells which in turn have an inhibitory effect on the immune system. So we are currently in the process of studying the function and the role of the ion channels and the regulatory T cells in the tumors but for now, for these papers, we mainly focused on the CD8 cells, which are very important. (56:49)
Question: Now our next question. Why did you not study KCa3.1 in TILs?
Ameet Chimote: Well, we do want to study that and it’s just something we want to do in the future. We started, as I said in the presentation, we started with Kv1.3 because its importance and its role in the calcium signaling, and we are currently conducting studies for the KCa3.1 function also in the TILs. (57:15)
Question: Have any A2AR modulators been tested for immunotherapy?
Ameet Chimote: Yes, indeed they have been. I mean, A2AR modulators are currently in the testing phase for—they're currently in the testing phase for, as an adjuvant along with the standard immunotherapy drugs and they have shown promising results in mouse models. And also recently, there was a paper that was published in which they have shown that using an A2AR modulator along with the CAR-T cells has also increased the efficacy of these CAR-T cells to penetrate solid tumors. (57:54)
Question: Now, what is the reason for KCa3.1 dysfunction in CD8+ T cells if there is no change in expression?
Ameet Chimote: Well, the reason for the KCa3.1 dysfunction could be due to many factors. First of all, the KCa3.1 function is calcium-dependent. So we want to see, we are currently conducting studies to see if there is any change in the calcium sensitivity of these channels. (58:25)
Secondly, the KCa3.1 channels, their function is dependent on a protein called as calmodulin, which confers the calcium sensitivity to these channels. So we want to also study, we are in the process of studying calmodulin in head and neck cancer patients to see if they are affecting the KCa3.1 channel function. (58:44)
And last but not the least, this function, like any other proteins, is also controlled by several kinases. So this would be some future studies that we want to undertake to see if there is any dysregulation of the kinases in these cells which can cause a dysfunction of KCa3.1. Thank you. (59:01)
Question: And Dr. Chimote, it looks like we have time for one more question. Will injecting KCa3.1 channel modulator prevent tumor formation in mouse models?
Ameet Chimote: Well, there are preliminary studies which have shown in vitro that this can affect the KCa3.1 channel function, but this is something we want to undertake in the future and we want to look at it. Thank you. (59:29)
Alexis Corrales: Thank you again, Dr. Chimote. Do you have any final comments for our audience?
Ameet Chimote: Well, thank you so much to Gibco for giving this opportunity.
(59:39) – Slide 29
And I want to thank my mentor Dr. Conforti for giving, for encouraging me throughout this project and for giving me all the help needed for this. I also want to thank our lab mates and last but not the least, thank you to everybody who has been listening and who have given me wonderful questions to answer, and I still look forward to hearing your comments and suggestions and feedback, and thank you. (1:00:03)
Alexis Corrales: Before we go, I’d like to thank the audience for joining us today and for their interesting questions. Questions we did not have time for today and those submitted during the on-demand period will be addressed by the speaker via the contact information you provided at time of the registration.
We would like to thank Dr. Chimote for his time today and his important research. We would also like to thank LabRoots and our sponsor Thermo Fisher Scientific for underwriting today’s educational webcast. (1:00:30)
This webcast can be viewed on demand through March of 2019. LabRoots will alert you via email when it’s available for replay. We encourage you to share that email with your colleagues who may have missed today’s live event. Until next time, goodbye.
End Presentation: 1:00:51
Get to know Ameet:
Why did you choose cancer research?
I have studied medicine and worked as a doctor in India and I have seen from close (and personal) quarters how this disease can affect individuals and families. I have always been motivated to do patient-centric translational research which has an impact on understanding a disease process and which can lead to development of therapeutics to treat that disease. I want to understand the complexities of how cancerous tumors grow, I want to know why the immune cells cannot attack and destroy the solid tumors and I want to know what we can do to empower the immune cells to attack and destroy the cancer cells.
What motivates you to succeed in your field?
A challenge: For me, it gives me an opportunity to read in depth, come up with strategies to troubleshoot and often come up with “out of the box solutions” to solve the problem. I also love to master new techniques and adapt them to answer research questions.
Optimism. Even when experiments do not work, I do not lose hope. I keep troubleshooting until I am sure if the negative result is a true negative result or due to a technical shortcoming. I will then try to design the correct meaningful alternative experiment that will provide me with the required answer
Describe yourself with 3 words:
Honest, hardworking, humble.
On your days off, what do you do?
Relax, watch TV, gardening, testing new recipes in my kitchen!
What is outreach/STEM to you?
Working in an Academic University that encourages science and research, I have had the good fortune to mentor several young undergraduate students in our laboratory. It is very important to teach the new generation to be inquisitive, think critically, learn problem-solving approaches and I think STEM education is important to educate individuals in this matter. Furthermore, outreach of the work that we do in our research laboratories helps create awareness on how basic research is essential to bring about some breakthroughs in the field of medicine. I am also a big advocate that opportunities in research in the STEM field should be accessible widely and made available to everyone irrespective of where they come from and who they are. To encourage more diverse and deserving minds to pursue careers in STEM, I feel that there should not just be more outreach, but also more funding.
“Be fearless, learn from your mistakes and have fun”—this advice from my idol, the great Julia Child continues to inspire me in the kitchen and in the lab!
How do you relax after a hard day of work?
Nothing is as therapeutic as stirring a big pot of curry.
If you could convince everyone in the world to do one thing at one point in time, what would that thing be?
Be kind to one another and treat everyone with respect
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