“Mom, what if there is a cancer treatment that we already have but didn’t know that it treats cancer. Like, what if we just injected lemon juice into the blood of all cancer patients and that helped them. What if it was just that simple, lemon juice, and we don’t even know it yet”
I obviously didn’t understand the catastrophic effects of blood acidosis at age ten. A huge thank you to my poor parents, who enduring years of “what if” questions so that I may one day bother my postdoctoral mentor, Kristopher Marjon, with them.
At the beginning of the summer I had the incredible opportunity to travel to Stanford University through the generosity of the Frances Velay Science Fellowship Program. My goals for the summer included learning new scientific techniques and skills, contributing relevant scientific data to the field of immunotherapy and cancer research, as well as establishing a professional relationship with a renowned research institution. The Weissman laboratory not only has historic legacy as a beacon of early stem cell research, but continues to be internationally competitive in the scientific fields of pathology, immunology, immunotherapy, and cancer research. I am so grateful for this experience that was made possible by so many of those supportive of me at home, Haverford College, Stanford University, and the committee board of the Francis Velay Fellowship.
Saying that I’ve learned quite a deal this summer is a vast understatement of my time at Stanford University. The data that I have accumulated contributes to an ongoing research project that aims to better understand the cellular trafficking of a protein known as calreticulin. It has become increasingly important to understand the location, signaling, trafficking, and possible transfer of calreticulin from one cell to another because it has been shown to play an important role in maintenance of a cell’s life cycle and has the potential to be incorporated in cancer-immunotherapy.
In the never-ending struggle for homeostasis, there is a constant uphill battle for cells, tissues, and organs to maintain control over the many distinct and complex pathways and mechanisms. Specifically, there is a delicate balancing act between pro-phagocytic and anti-phagocytic signals, which serve to either to signal or sequester alerts to the innate immune system to phagocytose (engulf or destroy) another cell. The maintenance of these signals allows for regular and healthy programmed cell removal when a specific cell is either dysfunctional or no longer useful. If a cell is able to evade or manipulate these progressions, it runs the chance of developing into a cancer.
I think of a cancerous cell as a cell that is no longer apart of the organism’s “self”. For a cell to be apart of an organism, it must cooperate. A cell that is apart of the self must obey the signaling pathways and inherent mechanisms possessed by the organism, it must behave according to the systems of the body enforced upon it. A cell that belongs to the “self” must operate as the smallest part of “the whole” and must dedicate its entire being to helping the tissue, organ, and system remain healthy. Cancer cells do not do this, in fact, they individually select for destructive behaviors and compete against other non-malignant cells for resources. Not only can cancer recklessly proliferate within an organism, they are somehow able to do so under-the-nose of the organism’s immune system (Chao, 2011. The Weissman laboratory asks: What proteins or cellular interactions are normally in place to prevent cancer growth, how does cancer circumnavigate these processes to proliferate, and what can be done to reverse this process? These are just a few of the many questions the Weissman laboratory is looking to answer.
To answer some of the questions about how a cancer proliferates in an organism, it is important to first start with its means of communication. At Stanford University, there is no better protein to start with than calreticulin (CRT). CRT is most commonly recognized as a endoplasmic reticulum chaperone protein where it aids in the folding of other proteins produced by the cell. However, in a unique twist of events, CRT is sometimes trafficked to the surface of the cell and takes on the role of a cell surface protein. On the cell membrane, it acts as an “eat me” pro-phagocytic signal and recruits the help of the organism’s innate immune system to clear the cell that it decorates (Chao, 2010). This process of cell removal is a clever way for the cell to internally recognize that it needs to be cleared, or, as we will explore, transfer CRT to a cell that is in need of being cleared. The integrity of our body and our health depends on balancing “eat me” and “don’t eat me” signals to direct programmed cell removal. This allows for healthy cells to remain in the tissue to resume their normal function and, alternatively, destroying cells that have the potential to do harm.
This pathway is dependent on the fact that CRT is able to leave its job in the ER and take on a new role at the cell surface, however, the mechanism by which CRT is trafficked to the surface of the cell is not well understood. Even further, it has been suggested that CRT can be used by certain cells of the immune system to decorate other cells, allowing for the accumulation of CRT on the target cell’s surface to act as an indicator to the immune system to aid in its clearance of other cells. In order to understand the role of CRT in programmed cell removal we used a thioglycollate peritonitis model. Thioglycollate is used as an irritant and is injected into the peritoneal cavity of mice, which results in inflammation and induces recruitment of immune cells to the peritoneal cavity. In a study by Lagasse and Weissman in 1994,
Figure 1. Kinetics of neutrophils and macrophages in the peritoneum after thioglycollate injection. This data demonstrates the percentage of reactive cells in the peritoneal cavity at time 0 hours (the point of injection). Over the next four hours, the reactive neutrophil population (the first responders of the immune system that react quickly to an infection) climbs from 0% to ~65%, until about hour 5, but then falls steadily from ~65% to 0% over the next 19 hours. The reactive macrophage population at time 0h is relatively high because of the resident macrophage population, which is the predominant immune cell that is within the peritoneal cavity. After injection with thioglycollate the peritoneal cavity is overwhelmed by neutrophils and therefore the macrophage population drops dramatically by 4 hours. Then, from time point 4 hours to 24 hours, the reactive macrophage population climbs from about ~10% to ~60% as recruited monocytes mature into macrophages which is occurring at the same time the neutrophil population is falling by being phagocytosed by the macrophages. Figure adapted from original publication (Lagasse et al 1994).
it was demonstrated that the percentage of reactive neutrophil and macrophage populations in the peritoneal cavity of a mice injected with thioglycollate (an agent to produce peritonitis, inflammation of the peritoneal cavity) had similar kinetics. This finding was partially expected as neutrophils are the first responders during an inflammatory insult but are typically clear by the macrophage populations because they undergo apoptosis rapidly. The fundamental finding from this study is that mice overexpressing BCL2 in the neutrophil population (bcl-2) had similar clearance rates compared to control mice within the peritoneal cavity that was independent of apoptosis. Lagasse’s findings are critical in establishing a clear inverse relationship between the two cell populations, and indicated that there was a mechanism or switch that occurred that was independent of apoptosis that induced the removal of the neutrophils from the peritoneal cavity. These findings were some of the first demonstrations of program cell removal and have led the Weissman Laboratory to their most recent area of interest. Unknown to the research team at that time was the role of calreticulin and other molecules that mediate “eat me” and “don’t eat me” signals to the macrophages.
Revisited by Stanford University in 2016, a similar relationship emerges between cell surface levels of CRT and the time course of this experiment. Quantified flow cytometry, Figure 2 shows CRT surface levels from macrophages (the red line) drops dramatically from a mean fluorescent intensity (MFI) of 1000 to ~500, while the MFI of surface CRT on
Figure 2. Cell surface levels of calreticulin on thioglycollate recruited neutrophils and macrophages. Neutrophils over expressing BCL2 (PMNs) and macrophages (MAC) were isolated from the peritoneum at indicated time points after thioglycollate injection. Cells were stained for surface markers to distinguish between PMNs and macrophages and analyzed for cell surface levels of calreticulin by flow cytometry.
the neutrophils over expressing BCL2 (the black line) rises from about 300 to 1000. In summary, these two experiments shows that the dramatic rise of the neutrophil population at the site of infection is followed by the rise in macrophages, which then phagocytose the neutrophils. And, in addition, the neutrophils arrive at the site with relatively low cell surface CRT levels but accumulate a high cell surface level of CRT right before they are engulfed by the macrophages. Conversely, the macrophages arrive with high levels of cell surface CRT but have much lower levels by the time they start the phagocytosis process. Not only does this data imply an association between CRT transfer from the macrophage to the neutrophil, but it also points to CRT as being an important pro-phagocytotic signal independent of apoptosis.
At the beginning of my time at Stanford, all of these experiments had been previously performed in an in-vivo model. While this is an excellent model to utilize and aid in our understanding of the role of calreticulin in programmed cell removal, it does not allow for further genetic manipulation or convenient means of interrogating and investigating of the different cell populations and trafficking of calreticulin. For these reasons, my summer project was focused on recapitulating the in-vivo findings by using laboratory cell lines. This would allow the Weissman laboratory to interrogate at multiple levels and have higher fidelity in understanding the role of calreticulin in programed cell removal. The cells that I incorporated into my research were immortalized mouse macrophage cells, known as J774, forced to over express CRT tagged with mCherry. mCherry is a fluorescent protein that can be utilized to indicate where the cellular localization of the protein (in this case calreticulin) is located. Forcing higher levels of CRT fused to the mCherry protein creates a built-in tracking device for CRT. So, wherever mCherry was observed and expressed in the cell, it could be assumed that CRT was located there as well. Instead of neutrophils, the target cells that were used in my assays consisted of an immortalized human colon cancer cell line known as SW620. The SW620 cell line gave a surface to which the CRT-mCherry was able to bind when being co-incubated with the J774-CRT mCherry cells. My assays were able to be quantified for total and surface levels of CRT by flow cytometry, location of CRT and other proteins of interest with immunofluorescence imaging, and possible soluble CRT in the supernatant of our co-incubation experiments by enzyme-linked immunosorbent assay (ELISA).
Figure 3. Transwell assay. Target cells are placed on the top well separated from the bottom chamber by a porous (0.4 μm) filter. The bottom chamber contains the macrophages.
The transwell assays that were performed consisted of the two populations of cells, J774 CRT mCherry macrophages and SW620 colon cancer cells, configured like the image in Figure 3. The J774 CRT mCherry cells populated the bottom of the wells (shown in blue) and the SW620 cells sat on top of the filter (shown in red). Both cell populations were separated from each other by a 0.4 um filter through which only the supernatant bathing the cells and small proteins, such as CRT, could pass. Since it has been previously suggested that macrophages can decorate target cells with CRT, I was primarily looking for a transfer of CRT-mCherry from our J774-CRT mCherry macrophages to the SW620 cancer cells. Figure 4 shows an averaged trend of CRT on the cell surface of the cell populations. There is a trend showing a decrease in MFI for CRT-PE from J774 CRT mCherry macrophages alone or co-cultured with the SW620 cancer cells (solid red bar) to
Figure 4. Calreticulin transfer from macrophages to target cells. Macrophages were cultured on the bottom well, expressing calreticulin fused to mCherry, in the presence or absence of SW620 cells. Cells were harvested from the wells then calreticulin was detected by assessing presence by mCherry signal or by antibodies against calreticulin and analyzed by flow cytometry.
J774 CRT mCherry macrophages that were co-cultured with the SW620 cancer cells (red/black patterned bar). Additionally, there is a trend showing an increase in MFI for CRT-PE in surface CRT from SW620 cancer cells not co-cultured with J774 CRT mCherry macrophages (solid grey bar) compared to the SW620 cancer cells co-culture with the J774 CRT mCherry macrophages (grey/black patterned bar). This suggests that our control J774 CRT mCherry macrophages have a greater amount of surface CRT compared to the J774 CRT mCherry macrophages co-cultured with the SW620s, supporting the hypothesis that the J774 CRT mCherry macrophages can transfer their CRT. Additionally, the control SW620 cancer cells, on average, have a lower amount of surface CRT than the SW620 cancer cells co-cultured with J774 CRT mCherry macrophages, suggesting that they are able to receive CRT from the J774 CRT mCherry macrophages.
We also wanted to determine if there was a difference in the calreticulin that was released into the medium during the coculture. Figure 5 demonstrates the levels of soluble CRT in the
Figure 5 Soluble calreticulin is generated by macrophages. Macrophages were cocultured alone or in the presence of SW620 cells. Supernatant was harvested and tested for the presence of calreticulin by ELISA.
supernatant from these transwell experiments, which were determined by ELISA. Measured in nano grams per milliliter, J774 CRT mCherry macrophages that were not co-cultured with SW620 cancer cells show a high level of soluble CRT in the supernatant, ~9.5-10 ng CRT/mL, while SW620 cancer cells not co-cultured with J774 CRT mCherry macrophages produce ~0.25-0.5 ng CRT/mL, a negligible amount. The difference of CRT in the supernatant between the experimental co-culture group and the control is accounted for by the transfer of CRT from the from the J774 CRT mCherry macrophages to the SW620 cancer cells. These data also highlight that macrophages readily make calreticulin and secrete it into their local environment.
Assessment of CRT localization was also an important part of my research project in the Weissman Laboratory. While CRT is an ER protein anchored by its KDEL ER retention sequence, it uniquely has the capability to travel to the cell surface. For most cells, it has been estimated that up to 95% of the cell’s CRT levels are contained within the cell, affording little on the surface. Therefore, I also sought out to determine the cellular localization of calreticulin and potential interactions within and on the cell in both primary cells as well as in cell lines that I incorporated in my research projects. Previous observations demonstrate that CRT is expressed at very low levels, if at all, in neutrophils and at a much higher level in macrophages. In an effort to investigate previous findings by the laboratory I used human primary macrophages (hMacrophage) and neutrophils (hPMN). hMacrophages and hPMN cells were stained with a rabbit polyclonal antibody against CRT and a biotinylated ligand that stains modified sugars. Staining for CRT aimed to identify cellular localization of this protein, while staining for modified sugars aids in determining all possible binding sites for CRT. Additionally, all cells were stained with DAPI to observe the nucleus.
Figure 6A shows a robust staining for CRT in hMacrophages within the cell, most likely located within the ER and vesicles. Figure 6B shows staining for modified sugars on the hMacrophages, revealing that a majority of possible binding sites for CRT are located on what appears to be the surface of the cell. There is little localization of possible CRT binding sites within the cell, opposite of what we see from the CRT staining.
Figure 6. Calreticulin and MSS stain on human macrophage cells. Human macrophages generated from peripheral blood mononuclear cells were stained fix, permeabilized and stained for Calreticulin (A) or a modified sugar stain, MSS, (B). Nucleus was counterstained with DAPI. Images were acquired using a LSM710 Ziess confocal microscope and all images were examined with a 63x oil immersed objective.
While the same staining platform was applied to hPMN, there were very different results. In contrast to the hMacrophages, there is a lack of CRT signaling as seen in Figure 7A and the signal for the modified sugar staining (MSS) appears quite robust in Figure 7B. This is an incredibly interesting finding since it further supports and validates the CRT’s path of cellular trafficking and transfer from a macrophage cell to the target neutrophil cell. The results of CRT and modified sugar staining from the hMacrophage and the hPMN cells show that the hMacrophage cells have an abundance of CRT and a deficit of CRT binding sites, whereas the hPMN (the hopeful CRT target cell) has an abundance of CRT binding sites but extremely low CRT levels. These immunofluorescence images confirm what previous research has shown
Figure 7. Calreticulin and MSS staining on human neutrophil cells. Human neutrophils were isolated from peripheral blood and fixed and stained for Calreticulin (A) and MSS (B) and nucleus was stained with DAPI. Images were acquired using a LSM710 Ziess confocal microscope and all images were examined with a 63x oil immersed objective.
Figure 8. BTK localization in macrophages. Peritoneal macrophages were isolated and fixed and stained for BTK and the nucleus was stained with DAPI. Images were acquired using a LSM710 Ziess confocal microscope and all images were examined with a 63x oil immersed objective.
Still, another question remains: How does the cell traffic CRT from the ER to the cell surface? Prior research done by the Weissman laboratory suggests that bruton’s tyrosine kinase (Btk) aids in the trafficking of CRT to the cell surface. Immunofluorescence staining of Btk on macrophages derived from mice have a diffuse staining profile as demonstrated in Figure 8.
To assess the relationship between Btk and CRT, a Proximity Ligation Assay (PLA) was used. This assay has incredible sensitivity because it is a fluorescent assay in which fluorescence will only be observed if two proteins of interest are in close proximity to each other, or otherwise co-localized. This is accomplished by using primary antibodies against CRT and Btk and then applying probes that have oligonucleotide chains (sequences of DNA). If the two proteins are approximately 30-40 nm apart, the oligonucleotide chains interact to form a single stranded DNA ring, which is then replicated through rolling cycle amplification and a pile of DNA that has specific repeating sequences is formed. Fluorescent tags can attach to these repeating sequences, creating a signal that is strong enough to detected and quantified through immunofluorescence imaging.
Figure 9 demonstrates a Btk and CRT staining with PLA. The points of red punctate on Figure 9B indicate areas that are positive for protein-protein interaction. It is interesting that most of these points appear to occur at a distance from the nucleus, suggesting that most of the interaction of CRT and Btk occurs close to the cell surface. There is a relatively low level of interaction signal even for an experimental group. However, this might be due to the fact that resident peritoneal macrophage cells derived from mice were used for this assay. If these peritoneal macrophages were stimulated with lipopolysaccharide (LPS) or from mice that were injected with thioglycollate to induce peritonitis, more punctate indicating interaction between Btk-CRT may be expected.
Figure 9. Colocalization of BTK and Calreticulin occurs in unstimulated macrophages. Proximity ligation assay was performed to determine if BTK and Calreticulin colocalize together in unstimulated macrophages. Cells were fixed and permeabilized and PLA assay was carried out as described by the manufacture. Cells were incubated with secondary antibodies only (A) or with antibodies against BTK and CRT (B). Images were acquired using a LSM710 Ziess confocal microscope and all images were examined with a 63x oil immersed objective.
However, just observing our proteins of interest with immunofluorescence imaging isn’t enough to determine their location. In order to gather more definitive evidence, the last piece of my project at Stanford University attempted to optimize staining for CRT and modified sugars against different biomarkers. The purpose of this experiment was to take a biomarker for the cell surface, vesicles, ER, or other organelle and cross stain it with CRT or our modified sugar stain (MSS). If there is overlap between the two stains, such as the CRT protein and a cell surface biomarker, we can be confident that our images are really displaying CRT inhabiting the cell surface. Many of the stainings produced spectacular images, such as that in Figure 10. J774 parental macrophages were stained with a panel containing rabbit polyclonal antibody against CRT, phalloidin which stains actin which aids in deciphering what is intracellular, and DAPI to visualize the nuclei. In the unstimulated macrophage cells, it is apparent that most of the CRT is within the cell. There is, however, some cross-over between the phalloidin stain and the CRT stain and some staining for CRT outside of the phalloidin stain suggesting staining at the surface of the cell, confirming that CRT is observed on the cell surface.
Figure 10. CRT cellular localization in Macrophages. Macrophages were fixed, permeabilized and stained for Calreticulin, phalloidin and the nucleus was stained with DAPI. Images were acquired using a LSM710 Ziess confocal microscope and all images were examined with a 63x oil immersed objective.
A similar stain was done for possible CRT binding sites by staining J774 parental cells with our modified sugar stain (MSS), phalloidin, and DAPI in Figure 11. Some interesting differences arise between this staining panel and the staining that was performed against the CRT protein. There is a robust stain for our modified sugar stain (MSS) within the cell, what seems to be marking many vesicles and the ER. Additionally, there are more MSS punctate that are overlapping with the phalloidin stain and outside of it. This suggests that there are many possible CRT binding sites within the cell as well as on the cell surface. However, the catch-all clause for the MSS is that, while it picks up on all possible CRT binding sites, it also a lot of other binding sites that many other proteins besides CRT can also bind to.
Figure 11. Calreticulin cellular localization in macrophages. Macrophages were fixed, permeabilized and stained for Calreticulin, phalloidin and the nucleus was stained with DAPI. Images were acquired using a LSM710 Ziess confocal microscope and all images were examined with a 63x oil immersed objective.
As my my project winds to an end, I concluded that calreticulin is, in fact, a very tricky protein to study. My attempt to recapitulate findings of prior experiments done in the Weissman lab proved difficult when we did not see as robust of a transfer of CRT from our J774 CRT mCherry macrophages to the SW620 cancer cells. While this was disappointing, there are many reasons as to why this may not have been the best model to use. Both cells lines are immortalized, meaning that they have a much longer life span than most-derived cells, calling in question the normality of their pathways and mechanisms. Additionally, the cell lines used where mismatching species; the J774 CRT mCherry macrophages being an immortalized mouse cell line, while the SW620 cancer cells are an immortalized human cell line. Prior experiments used derived macrophages and neutrophils, while we were used two cancerous cell lines. All of these factors could have played a part in why the CRT transfer signal was not as robust as earlier experiments had shown, but, even the suggestion of transfer from these experiments gave insight into CRT’s trafficking abilities.
Interestingly, CRT was readily released by the J774 CRT mCherry macrophages into the supernatant bathing the in co-culture. This sparked many questions, such as, whether or not cell density was a factor (for either the macrophage or the cancer cells) to be taken into consideration while assessing secreted CRT. If, in future experiments, cell density proves to be a factor, then there would be an interest in investigating the possible mechanisms by which macrophages can assess their own population’s density and release appropriate levels of CRT. Or, conversely, how they may assess their target cell’s density and make important decisions on how much CRT to release per target cell. It is also unknown at this point whether secreted CRT is in anyway modified compared to CRT adherent to the ER or the cell surface, or, if our secreted mCherry-CRT actually retains the mCherry fusion protein. All of these details are important is understanding the nuisances of CRT export and transfer.
Aside from learning new microscopy techniques on some incredible equipment, I had success with assessing interesting patterns of CRT localization on primary human and mouse macrophages and neutrophils. It was great to know that what we were observing on the microscope held true to prior research and data when macrophages were observed with a high levels CRT protein and low levels of CRT binding site content, and neutrophils possessed a low CRT protein content and high levels of possible CRT binding sites. The assays for antibody staining experiments produced a large number of remarkable photos that visualize the relationship between a macrophage cell and a potential target, really breathing life into the CRT dependent relationship built between these two cell populations. These images were, and will continue to be, supported by cross examining with known, multiple biomarkers along with selective staining for the CRT protein or possible CRT binding sites to create the most definitive panel for CRT localization.
While assessment of co-localization of CRT and Btk by using the PLA has many more stages of optimization to undergo, it was incredible to quantify the closeness of these two proteins interactions. Being able to assess their co-localiztion in an unstimulated cell population, and still able to get a signal, only makes me more curious to know how much more CRT would co-localize with Btk under stimulated or stressed conditions.
It saddens me to conclude my time at Stanford University, yet I am excited for the future directions of this project! In the coming months I am hoping to hear back on additional projects such as the growth of different biomarker staining for CRT, MSS, and Btk, or PLA assays performed on primary cells or cells treated with LPS. I know the Weissman laboratory will continue to work towards answering the questions that they originally posed, as well as other questions that I have contributed along the way, such as: What innate mechanism does the cell possess in order to know to send CRT to the surface of the cell? What happens to the CRT’s KDEL signal once it leaves the ER? Does CRT gain new modifications or a new homing sequence in order to be exported? Are there other proteins or pathways involved in the trafficking of CRT? These questions, along with many others, are in amazing hands as I leave Stanford University to rejoin my fellow Haverford students.
I would like to give a final thank you to all who made my time at Stanford University a possibility. Thank you to Irv Weissman and his entire lab for being so welcoming; selflessly inviting undergraduate student researchers to get a taste an elite and competitive laboratory experience. Thank you to Dr. Kristopher Marjon, my post-doctorate mentor, who I tortured day-in and day-out and who should be rewarded for his patience and compassion. Thank you to my parents, grandparents, and other extended family who helped support me on this really incredible trip, as well as friends Marie Vastola and Yvonne Wilson. Thank you to Rachel Hoang and Tim Chaya, as well as the rest of the Haverford College KINSC faculty and department for investing an incredible amount of time and emphasis on the importance of summer research and creating the opportunities for all the summer KINSC scholars. And finally, thank you to the Frances Velay Summer Scholarship board of trustees, from whom I was very fortunate to receive funding which covered the expenses that would have prevented me from experiencing Stanford University. Their dedication to supplying young women with the tools needed to grow and cultivate a career in STEM and research is inspiring, as is their mission to continually hold the life of Frances Velay in memory.
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