Week 4: Clinic, Clones, and Injections (Oh my!) - Alexandra Kot

Alexandra Kot | Dr. Sandra Demaria

2026.06.22:

Back to the clinic on Monday morning with significantly more cases than were seen last week. It is a slow process to get used to the histology slides, but compared to last week, I would like to believe I am getting closer to recognizing actual structures within the tissues as opposed to just passively observing.

What I really appreciate about Dr. Demaria's approach is her dedication toward putting together the patient story before allowing the resident to make any conclusions about the slide. She makes an effort to understand the motivation behind a lumpectomy, a biopsy, or a mastectomy and compares everything to past patient experiences, other imaging like breast radiographs, and family history until she is able to see a full story. It is very evident that she is giving all patients ample attention and treatment. 

One particular patient stood out to me. She had received a modified radical mastectomy after some neoadjuvant therapies. However, noted in her chart was how she had experienced cancer-related breast changes but, due to her fear of doctors, had waited before having it addressed. The cancer ended up being HER2 positive, a notably aggressive and highly proliferative cancer. More unfortunately, she had received three different courses of neoadjuvant therapies before landing on a treatment course that did not end with progression of disease (TCHP->Enhertu->Herceptin, tucatinib, and capecitabine). While HER2 positive cancers generally respond well to such treatments, with a 92.2% 5-year survival rate (1), that statistic is dependent on early diagnosis and treatment. It is unfortunate that this patient felt limited by her fear of doctors, and it led to a delay in diagnosis and treatment. 

2026.06.23:

Today for our weekly meeting we had a presentation on biomedical ultrasound from Dr. Jeffrey Ketterling. While this is a field I have not had much time to think about, the imaging quality Dr. Ketterling showed was quite impressive, especially at the speed at which acquisition was able to be taken. I have high hopes for the capabilities of the 3D and 4D ultrasound projects. What stood out to me the most was his vitreous floater work, which unfortunately now I am noticing more of when I am using the microscope. 

The second clinic day for the week was today. I am changing my approach to using some of my off-time to learn how to read breast histology slides. I have found some lovely online pathology resources from Massachusetts General Hospital (for example, this page on ductal carcinoma in situ: https://learn.mghpathology.org/index.php/Breast:_DE:_Ductal_Carcinoma_In_Situ) which goes well into the individual structures and outline the various ways these conditions can arise and what changes should exactly be looked for on the slides. I am going to summarize some of the information on these pages to make my own resource for some of the more common conditions that seem to arise during clinic.

Today was also a day for mouse peritoneal injections with the CTLA-4 immune checkpoint inhibitor. These mice are either receiving no treatment, just radiotherapy (8 Gy), just CTLA-4, or a combination of radiotherapy and CTLA-4. CTLA-4 inhibitors block a pathway that prevents the overactivation of the immune system, therefore allowing T-cells to recognize and attack cancer cells (2).

2026.06.24:

Today was a mouse-measuring day so we can track the tumor growth/shrinkage changes as they respond to the various treatment arms being looked at in this experiment. Interestingly enough, some tumors in untreated mice were shrinking while others, which were receiving both radiotherapy and CTLA-4 inhibitor, saw tumors that had continued growing and had begun to necrose due to their size and level of protrusion from the skin. We are unsure as to why the tumors are reacting this way. 

From the single-cell clones I plated on Friday, I was finally able to locate wells that contained just one cell. As we cannot guarantee that both cells (if two cells ended up being dispensed) have the MLKL or MAVS knockout, it is very important to confirm that only one cell is present in each well before expanding the knockout cells. Such an example of a group of cells from one cell is shown below in Figure 1. 

Figure 1: Single-cell cloning. A: 96-well plate under a microscope, with circled wells. B: One island of cells, which had expanded from one TSA cell. 

2026.06.25:

I aided in running a western today. Absolutely not my favorite one to do, but I understand the importance. Especially considering I will have to run westerns to confirm the genetic circuit output for my thesis project.

Cells are continuing to grow larger. A considerable number were identified as being grown from one cell, which is fantastic. I will check in on them again tomorrow to see if they are ready to expand. Otherwise, I can wait until Monday. 

2026.06.26:

More mouse measuring today. Maxine went deeper into the proper scruffing technique (Figure 2) to restrain the mice so that their tumors can be measured to track any changes potentially based on different treatments. Interestingly enough, some of the tumors, which were measuring around 3 mm lengthwise, managed to disappear since we last measured the tumors on Wednesday. 

Figure 2: Mouse work. A: Brown mouse, very cute. B: Proper scruffing technique demonstrated by Maxine. The thumb and knuckle of the forefinger scrunch the skin behind the mouse's neck to create a transverse fold. The remaining fingers curl in, with the pinky finger capturing the tail of the mouse. C: TSA tumor measured using a caliper to estimate tumor volume. 

Made my first gel for a western! No bubbles and no spilling out. 

Figure 3: 15-lane, 1.5mm, acrylamide gel poured for a western blot

Not all of my knockout cells have grown to be large enough to culture today. So I will let them grow over the weekend and culture them on Monday. 

References: 
1. “Female Breast Cancer Subtypes - Cancer Stat Facts,” SEER. Available: https://seer.cancer.gov/statfacts/html/breast-subtypes.html
2. “Definition of CTLA-4 - NCI Dictionary of Cancer Terms - NCI.” Accessed: Jun. 25, 2026. [Online]. Available: https://www.cancer.gov/publications/dictionaries/cancer-terms/def/ctla-4

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