What doesn't kill you, makes you stronger - I think cancer takes that a bit too seriously, when it comes to exploiting body's own defences in its favour. It exploits the immune system; it exploits the embryonic developmental mechanisms; and it hijacks wound healing machinery.

Cancer hijacking the wound healing machinery
Illustrated by @scienceblocks, using the following images -
car by OpenClipart-Vectors and gangsterby mohamed_hassan.
CC0

Previously on wound healing

In my previous post, on dynamics of wound healing I told you about different phases healing and cellular players that are required during process. I described the immediate events that happens during healing. The first to come is hemostasis which stops excessive blood loss by vasoconstriction and by recruitment of platelets which forms the fibrin clot. This is followed by vasodilation and recruitment of immune cells. In the meantime, the epithelial cells in epidermis start to proliferate and migrate to close the would. Working hand in hand with epithelial cells are activated fibroblasts in the dermis, which degrades fibrin, and deposit the lost extracellular collagen matrix. They then hold tightly to fibers of newly deposited matrix and contract the wound accelerating the healing process.

However, sometimes during the healing process the fibroblasts fail to deactivate and die. This may happen due to chronic wound signals or repetitive injury or insult to the organ. This persistent activation of fibroblast leads to formation of hypertrophic scars and even fibrosis - which is hardening of the tissue causing loss of organ function. I described in my previous post, how fibrosis is either cause or consequence of many chronic diseases and how it is one of the major cause of death globally. I also mentioned that solid tumors are fibrotic tissues. Probably because they exploit the tissue repair and development machinery; and this is what we are going to look at closely in this post.

Cancer - the rebellion of cells

You think becoming a cancer cell is easy? You think the body of the organism will let you win so easy? I guess not. If you were a cancer cell, you are like this rebel - probably the one who wants to seize the means of production and then take over the world. But, you have this entire world against you. And, no you can't take the world by surprise, because they have always anticipated a rise of such rebellion. They have defences against you, built during millions of years of evolution, ready to be deployed.

You thought, a single mutation will help you multiply uncontrollably. Well no you have a hell lot of intracellular checkpoints in place. You mutate one of the oncogene, you have a tumour suppressor gene in place. You mutate a tumour suppressor gene to escape cell death, you have redundant mechanisms to take care of it. It is now well recognised that you need multiple genetic insults to create the first tumour initiating cell (Chial, 2008). In fact, as we talk, there will be at least 1 new mutation that gets stabilised in one of your cells. By the end of the day there will be a lot of cells in your body with at least one new mistake (Risques and Kennedy, 2018). If you look closely in your gut you will find that upto 37% and 80% cell accumulate TP53 and Notch1 mutation respectively (Martincorena et al., 2018), and yet it's not necessary that you get a tumour, let alone cancer.

Even, if some set of mutations and epigenetic changes causes the tumour, you still need much more than just dividing like crazy to cause cancer. The soldiers of body, aka immune cells are going to treat you like a felon and kill you. So you need to fool them or corrupt them in your favour. You need to become more notice, travel through the body. You need to spread your business to multiple tissues to become cancer. So basically you need to have the environment of body in your favour. From a local goon, you need to become an inter-tissue mafia Don, with network all over the body.

What kind of environment exists in body where cells are allowed to multiply and immune system aids their growth and migration? Well, one such environment wound healing process. If a tumour cell can make use of such a process in an exaggerated manner, it's in business.

Exploitation of wound healing machinery

A solid tumor is an overhealing wound, is not a new idea. As early as 1863, Rudolf Virchow, proposed that chronic irritation and previous injuries sites in the tissue, favours tumorogenesis (Schäfer and Werner, 2008). In fact, it is known that both persistent inflammation of the tissue and injury sites are hot spots for fibrosis and tumors. In 1972, Alexander Haddow, molecular drew parallels between wound healing and carcinogenesis. Then in 1986, in publication Tumors: Wounds that do not heal, Harold F. Dvorak showed how anatomy of tumor resembles that of a fibrotic tissue or an overscarring wound. That is to say that any solid tumor is an epithelial tissue, surrounded by fibrotic connective tissue called - tumor stroma.

So the basic difference between a perfectly repaired wound, a scar, a fibrotic organ and tumor, lies in extent of exploitation of wound healing machinery. In a regenerating organ, you heal via bringing the tissue back to their initial state. In an imperfect repair as we usually witness on our skin, healing ends in remodeling phase, leaving a tiny bit a hypertrophic scar behind. However, when then fibroblast cells that remodel the matrix of any tissue during healing, remain persistently activated, you get fibrosis. And when the fibrosis is caused by rebellion epithelial cells, that wants to divide and conquer the body, you get a tumor.

Nevertheless, that's the bigger picture. But the main question remains that how is the machinery of wound healing being exploited by the cancer cells. To understand that we can revisit the wound healing phases once again and see how the different phases of normal healing process are exaggerated in cancer.

Phases of wound healing and their exploitation by cancer

Wound healing phases
Häggström, Mikael (2014). "Medical gallery of Mikael Häggström 2014". WikiJournal of Medicine 1 (2). DOI:10.15347/wjm/2014.008.| Public Domain.

If you remember from the previous article the wound healing happens in following overlapping phases - stopping the blood loss by vasoconstriction and clot formation (hemostasis), recruitment of immune cells (inflammatiory phase), expansion of cellular pools (proliferative phase), migration and explansion of epithelial cells (re-epithelialzation). In the dermis there is migration, proliferation and recruitment of fibroblasts, which secrete extra cellular matrix (ECM) and causes contraction of wound. This is finally followed by remodelling phase, where fibroblasts try their best to restore the mechanical properties of the matrix to initial state. The carcinogenesis also borrows the molecular machinery of these phases, for different purposes, as you will see.

The fibrin clot and tumor

The tumor cells, as they start growing and spreading, release factors that resemble those secreted during wound healing. These also includes enzymes that destroys the nearby tissue; and recruits immune cells such as neutrophils and macrophages that clears up the debris. But this destruction also leads to healing response that causes fibrin clots to form, which keeps getting replaced by collagen. But, presence of fibrin clots in tumors is prominent. In fact, this makes fibrin clots a target for early detection of tumors (Obonai et al., 2016). The difference, however, lies in the fact that - during wound healing clot formation stops a few hours post wounding, but it case of tumors it keeps forming in tumor stroma. The exploitation of clot formation machinery by tumor cells, may also be related to frequent incidence of thrombosis, found in cancer patients.

Making friends with immune cells

Image showing some of the mechasims employed by cancer cells to evade being killed by immune cells, and instead using them for their selfish goals
Image by Fronteirs in Oncology | CC BY 3.0

For most part our immune system is equipped to recognise and destroy the cancer cells. But not if cancer cells adapt to be friends with them and use it for their own benefit. For instance expressing NFkB, a transcription factor that promotes inflammation, also provides survival advantage to the cell expressing it. Moreover, NFkB expression promotes proliferation of cells and angiogenesis (blood vessel formation) in nearby tissue. That is the case of injury. In case of cancer cells which also has constitutively active NFkB, it also helps in promoting metastasis of cancer cells. (Gambhir et al., 2015).

When we have an injury or infection, usually the first immune cells to report at the site, are neutrophils. Well, even solid tumors are loaded with neutrophils. In fact, the tumor cells secrete a factor called G-CSF, which bias the bone marrow to produce more myeloid cells, including neurtrophils. Neutrophils, not only aids in formation of tumor stroma and growth of tumor, but also in its spread. If you remember, then in previous article I talked about, how neutrophil causes some collateral damage in neighborhood near the wound. The cancer cells makes the best of it and uses the destruction by Neutrophils to spread more efficiently, and keep the chronic injury going. It has also been observed that secondary site of metastasis, the Neutrophils proceeds the secondary tumor formation. But, doesn't it make you wonder that, how come, Neutrophils which are supposed to kill the cancer cells are helping it grow? Turns out, that not only tumor cells have resistance to neutrophil induced damage, but they also program neutrophils in specific way (for example, see my previous blog and Singel and Segal, 2016).

Immune cells that follow neutrophils at wound site are macrophages. The phenotype of tumor associated macrophages also resemble that of M2 macrophages found at the wound site. The poor macrophages in the tumor site, spend their life thinking they are healing an injury, but, in reality they are promoting tumor progression in a wound that would never heal (Muluaditan et al., 2018).

The late responders at wound sites are lymphocytes. The lymphocytes, esp T cells, also infiltrate tumor site. Now, T cells are very good at fighting the cancer cells. In fact, presence of T cells at the site of tumor, predicts positive prognosis. But, successful cancer cells are smarter than that. They downregulate their expression of MHC II, to avoid detection via CD4+ helper T cells. They use the gamna-delta T cells in their favor, and explore their production of IL17 and G-CSF. The persistent presence of neutrophils, inhibits the cytotoxic T cells activation at the site. Moreover, the cancer cells favours the recruitment of FoxP3+ regulatory T cells (TRegs), instead. The TRegs further inhibits the T helper and cytotoxic T cells from doing their job Singel and Segal, 2016, Beyer and Schultze, 2006).

In is now know that cancer associated fubroblasts (you will meet them soon), produces a protein called CXCL12, that recruits T lymphocytes, like it would in the wound. However, some of the fibroblasts makes an isoform of CXCL12 instead, called CXCL12-beta. This isoform is reponsible for recruiting T-Regulatory cells, and they even increase their survival (Givel et al., 2018).

The proliferative phase, re-epithelialization and metastasis

During the normal healing process the stem cell niches are activated. The epithelial cell numbers expand via rapid proliferation. So, does in cancer. Just that cancer cells don't have an off switch. The cancer initiating cell gives rise to rapidly proliferating proginitor cells. The cancer initititing cells maintains it's niche, just like stem cells would (Arnold et al., 2014). These cancer stem cells also initiate inflammation, which act as a feedback loop to hijack the wound healing pathway.

The downregulation of caspase8, is an important event in initiation of wound healing response. In 2009, Jamora lab, demonstrated that knocking out caspase8 is enough to recapitulate all the phases of wound healing in entire skin of the mouse. A similar downregulation of caspase8 in epithelial cells of carcinoma, is also hallmark of many tumors.

Another common signature of wound healing and cancer is activation of transcription factor Snail/Slug. In our lab we find overexpressing snail in epidermis of mice recapitulates wound healing, causes dermal fibrosis, and make mice highly likely to get squamous cell carcinoma (Du et al., 2010). Snail/Slug also increase survival of cancer stem cells. It also makes them more fibroblast like and hence more motile. Hence, Snail aids in metastasis.

Moreover, re-epithelialization of wound requires a two state switch. One that turns on migration of epithelial cells and one that turns it off. Sundaram, 2017, showed that the migration is initiated by activation of FSTL1. The FSTL1 off switch is dependent of its mRNA degradation by microRNA, mir-198, during normal healing. However, epithelial cells of carcinoma, the mir-198 switch is missing. This helps the cancer cells to migrate uncontrabally and successfully breaching the barrier of their primary site.

Tumor stroma - hijacking the construction workers

By now, you must have realized the dependence of tumors on surrounding microenvironent. In fact the carcinoma cells itself contributed to about 30% of cells in tumor mass. The rest of the cells live in a stroma that feed the cancer cells and make things easier for them to spread, by trying to heal a non healing wound. But, one of the important aspect of wound healing process is healing of the dermal tissue. The connective tissue layer right below the epidermis.

The cells that live in this region are the construction workers called fibroblasts, whose usual job is to maintain the tissue integrity. The secrets the collagen matrix which holds the things together. When fibroblast detect the wound they migrate to the wound site, they get activated, secrete collagen in excessive amounts and become myofibroblasts that the contract the wound.

The fibroblasts that make the tumor stroma, resembles the activated fibroblasts or myofibroblasts, found in the wound. However, unlike wound the fibroblasts in tumor stroma are fibrotic. Which means, that they are perpetually activated. Which is why solid tumors are also thought of as fibrotic tissues.

Anyhow, one of the hot questions in the field however remains origin of these cancer associated fibroblasts and their functional hetrogenity (more on that later). The first candidate is the resident organ fibroblasts that cancer cells recruit to form tumor stroma. Adipocytes, pericytes, fibrocytes and mesenchymal stem cells are also considered candidates that differentiate into myfibroblasts that gets recruited at wound site and tumor. Recently even macrophages have been proposed to transdiffrentiate into myofibtoblasts found at wound site. However, it remains to be seen that whether this is also the case with tumor stroma. The other implicated source of myofibroblast in tumor stroma, but not in wounds is Epithelial to mesenchymal transition (EMT). Snail, which is commonly overexpressed and unregulated in many cancers, is known to cause EMT during embryonic development. No EMT has been observed in wounding. But EMT is commonly observed on in vitro studies using cancer cells. Nonetheless, the in vivo lineage tracing studies cast a doubt on EMT in cancer. EMT as source of myofibroblasts, hence remains a debated topic (see Sinha et al., 2018 and Shiga et al., 2015).

Summary

In a nutshell, what we saw in this discussion is that cancer hijacks wound healing machinery. It fools a lot of cells into trying to repair a wound that would never heal. A wound environment is quite favourable for growth and metastasis of cancer. Understanding tge simalarities and differences between a wound and tumor, provides an oppurtunity to make more efficient treatment strategies. Especially, strategies that targets tumor stroma.

As I describe my research further, we will bump back into this topic again. I think for today, this much information should suffice.

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References

1. The biology and physics of wound healing (part 1 - the cellular players and dynamics of healing)

2. Fibrosis - an overscarring wound

3. Chial, 2008. Tumor suppressor genes act as “brakes” to stop cells before they can travel down the road to cancer. A loss of function mutation in these genes can be disastrous.

4. Risques RA, Kennedy SR (2018) Aging and the rise of somatic cancer-associated mutations in normal tissues. PLoS Genet 14(1): e1007108. doi:10.1371/journal.pgen.1007108

5. Martincorena et al., 2018. Somatic mutant clones colonize the human esophagus with age

6. Matthias Schäfer & Sabine Werner, 2008. Cancer as an overhealing wound: an old hypothesis revisited

7. Alexander Haddow, 1973. Molecular Repair, Wound Healing, And Carcinogenesis: Tumor Production A Possible Overhealing?

8. Harold F. Dvorak, 1986. Tumors: Wounds That Do Not Heal

9. Obonai et al., 2016. Tumour imaging by the detection of fibrin clots in tumour stroma using an anti-fibrin Fab fragment

10. Gambhir S, Vyas D, Hollis M, Aekka A, Vyas A. Nuclear factor kappa B role in inflammation associated gastrointestinal malignancies.

11. Base calling neutrophils to destroy tumors!

12. Singel KL, Segal BH. Neutrophils in the tumor microenvironment: trying to heal the wound that cannot heal. Immunol Rev. 2016;273(1):329-43.

13. Muluaditan et al., 2018. Macrophages are exploited from an innate wound healing response to facilitate cancer metastasis

14. Regulatory T cells in cancer. Marc Beyer and Joachim L. Schultze, 2006.

15. Givel AM, Kieffer Y, Scholer-Dahirel A, et al. miR200-regulated CXCL12β promotes fibroblast heterogeneity and immunosuppression in ovarian cancers. Nat Commun. 2018;9(1):1056. Published 2018 Mar 13. doi:10.1038/s41467-018-03348-z

16. Arnold KM, Opdenaker LM, Flynn D, Sims-Mourtada J. Wound healing and cancer stem cells: inflammation as a driver of treatment resistance in breast cancer. Cancer Growth Metastasis. 2015;8:1-13. Published 2015 Jan 29. doi:10.4137/CGM.S11286

17. Lee et al., 2009. Dynamic expression of epidermal caspase 8 simulates a wound healing response

18. Du F, Nakamura Y, Tan TL, et al. Expression of snail in epidermal keratinocytes promotes cutaneous inflammation and hyperplasia conducive to tumor formation. Cancer Res. 2010;70(24):10080-9.

19. Sundaram et al., 2017. EGF hijacks miR-198/FSTL1 wound-healing switch and steers a two-pronged pathway toward metastasis

20. Sinha et al., 2018. Direct conversion of injury-site myeloid cells to fibroblast-like cells of granulation tissue

21. Shiga K, Hara M, Nagasaki T, Sato T, Takahashi H, Takeyama H. Cancer-Associated Fibroblasts: Their Characteristics and Their Roles in Tumor Growth. Cancers (Basel). 2015;7(4):2443-58. Published 2015 Dec 11. doi:10.3390/cancers7040902