Act One - Osteoid cells

New thought!

Instead of the pan up to reveal the tumour mass (seen in "Act One - Transition from prologue") after the prologue, there should be an intro of the osteoblasts and osteoclasts. The introduction won't be too detailed; we'll only mention that there is a balance achieved by the two cells types. Blasts do one thing, the clasts do another.

Figure 1 will be the scene that bridges the prologue and the first act. After this scene, we see the tumour mass, and I think after seeing the tumour mass, we would revisit the osteoblasts and clasts and see how their functions are altered.

FIGURE 1.

Talking to Dr. Mongeau yesterday clarified some of the questions I raised earlier about the appearance of osteoblasts when active and inactive. They are indeed spindle shaped when inactive, and they are virtually undetectable during this state. When active however, they balloon up to appear like a row of cuboidal cells. I chose to make them appear like translucent balloons. Note also the red coloured osteoclasts with multiple nuclei sitting in Howship's Lacunae... I definitely need to draw them larger next time!

FIGURE 2. Osteoblastic caviar...

I'm still not sure if they grow while mainting volume, or if they acquire volume when they enlarge. I figure people just... don't know... Hooray for artistic interpretation...!

Round table 3.5

In attendance: Dr. Mongeau who's a pathologist working at l'Hôtel Dieu in Sherbrooke, and Louis Doré-Savard who's a Ph.D student from Dr. Sarret's lab.

Dr. Mongeau showed up some of his slides and explained a few things to us about breast cancer, a subject he specializes in. (Gah... preposisitons...)

We talked about some of the differences found between lobular and tubular breast metatases, how the former is generally more diffuse while the latter grows as a cohesive mass. Lobular metastases may produce signet cells, cells that contain a large mucous vacuole with a nucleus that has been pushed aside, thus giving it a ring like appearance. Tubular metastases, if well differentiated, arrange themselves so that there are numerous ducts and tubules found within the mass.

On the metastatic process, I was reminded that lymph vessels play a HUGE part. How could I have forgotten! Sorry Dr. Taylor... Anyways, an interesting tidbit is that cancer cells, in his opinion, intravasate from their site of origin into NORMAL peripheral vessels. So, instead of entering a vessel created by tumour angiogenesis then migrating into a normal vessel, the cancer cells enter the normal vessels directly! And yes, they most likely enter as single cells, then agglomerating to create emboli later on.

Finally. About MUMMIES! Having been fed tons of visuals and animations where we see apoptotic cells crumpling and dissolving into nothingness like the Ring-wraith stabbed by Eowyn, it came to me as a surprise that necrotic cells don't necessarily lose their ultrastructure! I mean, duh, I should've known. If a cancer grows reaaally fast, and any cell at a distance greater than 1 mm from a nutrition supplying vessel cannot survive, then... well, the entire centre of the mass of tumour would simply die off and be caught within a shell of viable cells. Sorta like the dead organic center of a tree trunk. Only the outside is actively growing.

Anyways, these dead cancer cells become mummified in the sense that their cytoplasmic contents coagulate. Having coagulated, the cell sort of glazes over. On histological slides, therefore, these pomegranate-like cells have indistinguishable organelles because of their inability to stain properly. Well, I thought that was cool.

Round table 3

In attendance: Nicolas, Philippe, Sarret's lab.

During the recent weekly lab meeting held by Dr. Sarret and his lab, I presented my draft for the prologue, and I outlined how I imagined the breakdown of the rest of the animation would look like. We had a discussion, and I went back to the drawing board (aka Pages). So, here's a very bare-bones script. Soft tissue to come later...

ACT ONE

"Osteoid Pathophysiology"

We will begin showing osteoblast and osteoclast function. Then, introduce cancer factors that alter both cell types. Note, however, that osteoclasts will be the main protagonists/antagonists of the story.

We first start with those that alter osteoclasts directly. We begin to see increased activity and numbers. Then, we see osteoblasts being modulated by factors causing an increase in RANKL expression. This then also increases osteoclast activity.

An osteoclast steps into view, and we end up in its cytoplasm very briefly. We see vesicles and stuff, and protons being pumped out of the cell. Zooming back, we reveal the ruffled border of the osteoclast and see it in action. We then zoom out to see some damage. Exposed nerve endings, and acidic environment.

ACT TWO

"Nociception"

Exposed nerve endings are now susceptible to mechanical stress. Show how.

Inflammatory cells appear. They release factors. Follow the inflammatory cell to the cancer mass surface. See the cancer cells release their own factors.

Follow these factors to the nerve endings. Increased firing.

ACT THREE

"Acidosis and Peripheral Sensitization"

Activated nerves are further aggravated by acidic environment. See allosteric shift in receptors. Show this by following a few protons.

Elaborate on firing frequency and amplitude.

EPILOGUE

"Allusions to Central Sensitization"

One thing that I'm working out now, is that nerve fibres are found surrounding the vessels within the bone cavity. These fibres are in the marrow and lie close to the trabecular surface, but is not in it. On the other hand, the nerve fibres in the compact bone, become exposed only after the resorption process has started. The narration and flow of the current dialogue seems to imply that pain only occurs once the nerve endings in the resorbed bone is revealed.

Indeed, bone resorption occurs simultaneously as tumour growth itself. Temporally, therefore, the nerve ending within the bone may technically be responsible for initial pain symptoms. Nevertheless, I feel we are neglecting the fact that pain can occur first by other means. That is, nociceptor activation may first occur by compression of nerve fibres around marrow vasculature, and by the binding of cancer factors into the marrow cavity.

I feel I need to discuss this further.

Act One - Transition from prologue

Sketched some ideas for transitions from the prologue to the first act. Please excuse the fairy tale colours... I intend to make the final product... less saturated...

Figure 1 is the last shot from the prologue where we find ourselves amongst a trabecular forest. From this point, the camera zooms in and upwards. Cue HEART CLENCHING DRAMATIC MUSIC AND TITLE...!

FIGURE 1.

The next tryptic (Figure 2) is an idea of what the camera will show next. We continue to move upwards to reveal a cancer mass. There are chemokines being secreted, inflammatory cells are abound. (I also plan to include osteoblasts and osteoclasts lining the trabecular and some nerve endings.) Basically, this is a huge setting-the-stage type of shot. Please read Figure 2 from the bottom panel upwards.

FIGURE 2.

Prologue - scripts

Here are the tentative narratives that will accompany the prologues. Wording has been selected to best communicate with each audience. To begin, here is the one intended for the patient-oriented prologue (please refer to storyboard panels posted earlier in "Prologue - breast cancer metastasis")

BREAST CANCER METASTASIS

Fade in from black. Faint lighting reveals silhouette of woman. Camera is zoomed in and just above level of breasts. Lights and shadows being to undulate over skin surface as camera slowly pans downwards.

"A metastasis is a malignant development at a site apart from its origin. Patients suffering from cancers of the kidneys, lungs, and prostate have a chance of developing a metastatic tumour of 50% of higher."

Short pause.

"Breast cancer patients have an 80% chance."

Camera should be near level of hips, immediately above the head of femur. Undulation of shadows slow down and take shape of trabecular matrix.

"And the most common site is in bone, such as that of the thigh."

Trabecular matrix now well defined.

"A tumour implanted here will result in one of the most painful conditions a patient may ever experience."

And this next one will be the one designed for the scientific audience (please refer to the video clip previously shown in "Prologue - the metastasizing cell 2"):

THE METASTASIZING CELL

Start from black. Spot lighting reveals a growing tumour mass.

"Of the most excrutiating pains, the one induced by bone cancer ranks among the top."

Singular cancer cell breaks off.

"And this experience may start with a single cell. That has broken off from the primary tumour at the breast. And having intravasated into the ciruclation, is well on its way to its hosts' femur."

This cell is caught by a macrophage. Enter cancer embolus.

"This cell, like many others, does not make it."

Pause.

"But inevitably, some will. Such as these cells, traveling as an embolus, as they so often do. And, with their arrival to the bone matrix at the metaphyseal plate, a series of events are initiated that ultimately lead to pain."

Background dissolves to reveal trabecular matrix.

Prologue - the metastasizing cell 2

As promised, here's a new storyboard clip for the revamped prologue!

VIDEO 1.

In this one, I've opted to show a single cell breaking off from the main tumour mass again. However, it is caught by a macrophage. We follow a metastatic embolus from then on. By doing so, I will be able to suggest that intravasation of cancer cells may be active as well as passive. Also, I am able to show that cancer metastasis is still a very random and ambiguous process.

The red blood cells (RBCs) shown in this clip are like dead weight in nature. They start off lying on the ground. As the story progresses, however, the RBCs will pick up speed as they are literally swept up by the circulation. Near the end, they will slowly sink back to the ground. These differences in RBC suspension will respectively represent the states of circulation found in tumour vessels, normal arteries, and sinusoids within bone.

Tumour angiogenesis and metastasis

I've read up on tumour angiogenesis, and have learned that tumour vasculature is really a meshwork of unorganized tubular interconnections (Fukamura & Jain 2007). These vessels have varying diameters, numerous trifurcations, saccules, and they often don't have a continuous endothelial layer or basement membrane (Figure 1).

FIGURE 1. Normal (left) vs. cancerous (right) vessels.

Because of the disorganization of endothelium, tumour vessels have large fenestrations that may be up to 2 um in diameter (Figure 2). This, on top of numerous pores, makes these vessels very leaky. As a result, cancer cells can easily intravasate into these vessels because of the loose extracellular matrix, and the thin or non-continuous vessel walls. With cancer, the process of intravasation - which is normally an active process of MMPs and cytoskeletal rearrangement - may become a passive procedure where tumour growth extrudes and sloughs off tumour cells into the compressed vessels (Bockhorn et al. 2007). This occurs at regions where endothelial cells are missing, where tumour cells act as the lumen wall (Figure 3).

FIGURE 2.

As seen in Figure 2, the endothelial layer exhibits numerous pores and intercellular fenestrations, and endothelial cells often form long cellular processes (Hashizume et al. 2000). As mentioned in the previous paragraph, tumour vessels are easily compressed by the unhindered growth of tumours, resulting in their tortuous and irregular shapes seen in Figure 3. Blood flow in these vessels are significantly lower than their normal counterparts. This allows for pooling of blood. Also, the irregularity of the blood supply results in numerous regions of necrosis.

FIGURE 3. Cancer tastes like watermelon!

Cancer cells are continuously shed into the circulatory system. But, few ever implant successfully into the metastatic site. From the literature I've read thus far, I'm under the impression that while tumour cells travel as aggregates, they may still intravaste individually. I would imagine that the chances of survival singularly are slim, however. What I found interesting is that platelets may aid cancer cells in evading the host immune cells in circulation (Palumbo et al. 2005); fibrin may prevent NK cells from recognizing the circulating tumour embolus.

As for extravasation at the metastatic site, in metaphyseal bone... Sinusoidal vessels predominate within spongey bone. They are large vessels with flow at 30-folds lower than that of arteries (Bussard et al. 2008). Similar to tumour vasculature, sinusoid endothelial cells interdigitate to create a highly-fenestrated and discontinuous layer. The basement membrane and the adventitial layer, which consists of phagocytic cells, are both discontinuous as well.

As of yet, again I have not found solid evidence that migrated cancer cells need to immediately break down bone extracellular matrix to implant, although evidence shows that they are functionally able to (Yoneda et al. 1997). MMPs may be used to break through the vessel walls and what little extracellular matrix there is. Once expose to bone, cancer cells adhere to the bone surface by use of adhesion molecules such as VLA-4 and ICAM-1 (Bussard et al. 2008). After this step, growth may begin.

A revisit to my storyboard idea "The Metastacizing Cell" will be necessary.