Development of Mouse-Derived Organoid Lines from Fallopian Tube Epithelial Cells for High Grade Serous Ovarian Carcinoma Modeling

So yeah, we are currently working on developing novel models of high grade serous ovarian carcinoma, models which are syngeneic in the sense that the protocol outlines. We take the healthy fallopian tubes from healthy mice and then we derive them as organoids in the Petri dish and then we give them all the different errors or genetic modifications that we would usually find inside of a normal human version of the disease. Now, this is very cool because on one side, you can study all kinds of intrinsic factors of these tumors inside of the Petri dish as organoids, which are more realistic than traditional 2D cultures.

But the thing is that since there's syngeneic models, you can then take these organoids and then put them back into a mouse, and then see how the tumor actually develops and then interacts with the host. This allows us to have a much more holistic view of, you know, how a tumor and tumorigenesis actually happens, which we hope that is going to give us much better tools and a much better understanding of how this disease actually works and how to better fight it. To begin, obtain euthanized female C57 black 6J mice.

Remove the ovary and fallopian tube and transfer them into a sterile Petri dish containing DMEM, supplemented with penicillin streptomycin at four degrees Celsius. Use a dissecting microscope, fine forceps and scissors to remove residual fat from the ovary. Isolate the fallopian tube, including the infundibulum and part of the distal ampulla from the ovary and uterus.

Now transfer the cleaned tissue to a tube containing the complete digestion mixture. With a pair of fine scissors, mince the fallopian tube into pieces smaller than 0.5 millimeters. Then incubate the tube at 37 degrees Celsius for 40 to 50 minutes before performing organoid culturing.

To develop the mouse tumor model, set up five injections, each containing four million cells resuspended in unsupplemented Opti-MEM I, an ice cold basement membrane matrix. To prepare the injections, first expel air bubbles from the dead volume of a U-100 insulin syringe fitted with a 28-gauge needle using cold PBS. Then slowly load the syringe with 40 microliters of cells and keep the loaded syringes on ice until the mice are anesthetized.

Once ready for injection, place the syringes beside a heating lamp to allow the basement membrane matrix to warm and increase viscosity, aiding injection and engraftment. Next, place a mouse in a prewarmed cage under a heating lamp to prepare for surgery. Make an incision less than one centimeter along the flank, above the thigh of an anesthetized mouse.

Cut through the epidermis and hypodermis to reach the bursa region. Gently pull out the ovary by locating the attached fat pad. Then using a prepared syringe, inject cells through the fat pad and ovary into the oviduct sac.

Carefully return the ovary into the bursa. Close the muscle layer using non-braided sutures. Subsequently, close the skin incision using surgical staples.

Place the mouse back in the warmed cage and maintain body temperature with a heating lamp until semi-conscious, usually for more than 15 minutes. Organoids derived from murine fallopian tube epithelium have a well-defined spherical and clear shape after about five days and require packaging around day 10 to 12. The wild type organoids are transformed into precancerous tumoroids using genetic engineering techniques, such as lentiviral or retroviral gene overexpression.

Single clone cell lines derived from the genetically modified organoids can be further studied in vivo by injection into the ovarian bursa. Tumors typically reach a size of two to three centimeters around 60 days post-injection. Well, we say that really, as I indicated before, like the biggest advantage that, you know, the way that this protocol works, of course, is to generate these syngeneic models, right?

And so since they're syngeneic, as I said before, you can really appreciate the whole interaction between, you know, a complete animal, a complete mouse, you know, and the tumor that you're developing while having, you know, like complete control over the genetics of the tumor. And so, of course, you know, compared to other models where maybe you're going to be trying to understand the immune system inside of the Petri dish, of course, it's going to be very difficult to replicate the immune system in the Petri dish. Or if you take PDXs, so like samples, tumor samples from patients that then you put into mice, in order for that to work, the mice cannot have an immune system or else it's going to reject this tumor, right?

But since this model is syngeneic, you can really appreciate the whole interaction of the whole host with the tumor and particularly with the immune system, which right now is one of the hottest areas of research in the field. So I really think that that is the most important benefit of this protocol compared to other protocols that you find elsewhere. So actually, we've been using these models already to investigate some very interesting phenomena.

I would say the one that I'm the most particularly interested right now is how the adaptive immune system, from what we've seen, seems to be a major contributor in shaping the genomic properties, particularly copy number variations of high grade serous ovarian carcinomas in the sense that if the immune system is not there, it seems that actually these tumors do not develop such massive and complex DNA rearrangements. And so we're trying to figure out how does this happen and how we could then hopefully try to turn it around in order to potentiate any kind of immune therapies against it.