Progenitors from the central nervous system drive neurogenesis in cancer

Welcome to our Monthly Journal Club! Each month I post a paper or two that I have read and find interesting. I use this as a forum for open discussion about the paper in question. Anyone can participate in the journal club, and provide comments/critiques on the paper by leaving a comment below. I picked this month’s paper because if these findings are true, it is a real paradigm shifting piece of work, substantially challenging what we know about cancer (and specifically prostate cancer), the nervous system, and cell migration in general. Also, this research falls squarely into my wheelhouse, as I too work on how the nervous system and cancer communicate! It was published in Nature, and is titled “Progenitors from the central nervous system drive neurogenesis in cancer” by Claire Magnon & colleagues at the François Jacob Institute of Biology in France.

Tumors interact with their local environment and by extension, the whole organism . These interactions can result in deleterious outcomes for patients, like tumor progression, metabolic problems, anorexia, inflammation, and sleep/circadian disruption. Magnon and colleagues provide evidence that in addition to these established pathways, neural progenitor cells leave the brain and migrate to the tumor (in a model of prostate cancer), promoting cancer growth and progression. (credit:   Walker II & Borniger, 2019   ) .

Tumors interact with their local environment and by extension, the whole organism. These interactions can result in deleterious outcomes for patients, like tumor progression, metabolic problems, anorexia, inflammation, and sleep/circadian disruption. Magnon and colleagues provide evidence that in addition to these established pathways, neural progenitor cells leave the brain and migrate to the tumor (in a model of prostate cancer), promoting cancer growth and progression. (credit: Walker II & Borniger, 2019).

The primary claim in this paper is extraordinary, and I quote it here verbatim: “Here, we reveal a process of tumour-associated neo-neurogenesis in which neural progenitors leave the subventricular zone (SVZ) and reach—through the blood— the primary tumour or metastatic tissues, in which they can differentiate into new adrenergic neurons that are known to support the early stages of the development of cancer”.

Neural Progenitor Cells    (DCX+, green), Astrocytes (GFAP+, blue), and blood vessels (CD31+, red) in the mouse olfactory bulb. These cells are born in the subventricular zone, and then migrate to the olfactory bulb along the rostral migratory stream to integrate into olfactory (smell) circuits. (Credit: CC; Oleg Tsupykov).

Neural Progenitor Cells (DCX+, green), Astrocytes (GFAP+, blue), and blood vessels (CD31+, red) in the mouse olfactory bulb. These cells are born in the subventricular zone, and then migrate to the olfactory bulb along the rostral migratory stream to integrate into olfactory (smell) circuits. (Credit: CC; Oleg Tsupykov).

This raises many many questions, like…how do newly born neural cells know how to get all the way from the brain to a tumor so far away? How do these cells make it past the blood brain barrier (BBB), and even if they do make it this far….how do we know that the cells that make it to the prostate are the same ones that left the brain? There are, after all, neurons in the peripheral nervous system (PNS) that could be infiltrating the tumor to cause these effects. The idea that newly born neurons can leave the nervous system is pretty wild on its own…but the claim that they not only leave, but migrate all the way to a distant tumor where they promote its growth is amazing….if true! Below, I’ll go through the primary figures in this paper one by one and explain what the authors are showing. If I feel like something is missing, or there could have been additional work done, I will say so. So far…it feels like this paper has not gotten the attention it deserves…probably because neuroscientists rarely talk to cancer biologists!

The authors started by looking at prostate tumor samples to see if they indeed contain neural progenitor cells. To label these types of cells specifically, they applied antibodies against doublecortin (DCX) which were tagged with a green fluorescent molecule. This way, all the DCX-expressing cells (i.e., neural progenitor cells) appeared green under a microscope. As one marker is not enough to convince the editors at Nature, they showed that these cells also express other markers of immature neurons (PSA-NCAM, internexin), but not markers of mature neurons (neurofilament-heavy (NF-H)) or epithelial cells (Pancytokeratin (PanCK)).

Figure 1: Neural progenitors (DCX+, PSA-NCAM+, INA+) are found in prostate tumor samples and they provide a prognostic indicator of cancer recurrence/survival.    These neural progenitors do not express markers of epithelial cells or mature neurons, and increased amounts of these cells within the tumor is associated with high-risk tumors compared to low-risk and benign (BPH) samples. Additionally, with each part of the prostate that the tumor invades, there is a concomitant increase in neural progenitor cells. (Credit: Mauffrey et al., 2019)

Figure 1: Neural progenitors (DCX+, PSA-NCAM+, INA+) are found in prostate tumor samples and they provide a prognostic indicator of cancer recurrence/survival. These neural progenitors do not express markers of epithelial cells or mature neurons, and increased amounts of these cells within the tumor is associated with high-risk tumors compared to low-risk and benign (BPH) samples. Additionally, with each part of the prostate that the tumor invades, there is a concomitant increase in neural progenitor cells. (Credit: Mauffrey et al., 2019)

After showing that these ‘central progenitor’ cells can be found in human tumors, the researchers moved to working in a mouse model of pancreatic cancer to more finely understand how these cells get to the tumor and what they do when they get there. First, though, I want to highlight that the marker that they use (DCX) to distinguish newly-born neurons is also expressed in the peripheral nervous system, which raises concerns that their interpretation of cells traveling from the brain to the tumor might be incorrect. Using a triple-transgenic strategy, they engineered mice to express enhanced yellow fluorescent protein (eYFP) in cells that make a human version of DCX (DCX-eYFP mice). This way, all DCX-expressing cells show up as yellow under the microscope (see Fig. 2 below). In addition to these genetic manipulations, mice were engineered to express myc, a proto-oncogene that is highly expressed in most cancers, specifically in the prostate, causing mice to develop prostate tumors similar to those found in humans.

When they analyzed DCX-eYFP cells within the brain and prostate of mice with and without tumors, they found eYFP+ cells in known brain locations, but only in the prostates of mice with prostate cancer. This suggests, that these cells are somehow recruited to the prostate during tumor formation, but not during normal functioning of the pancreas. A benefit to having cells that are labeled yellow is that we can easily run them through something called a fluorescence activated cell sorter (FACS; flow cytometer). This lets us label them with additional colors to see what other proteins they express, and quantify them with single-cell resolution. Using this technique, the researchers demonstrated that DCX-eYFP neural progenitors in the prostate do not express mature cell lineage markers (i.e., they are lin-negative), boosting the idea that these cells truly are progenitor cells (see Fig. 2b).

Figure 2: In a mouse model of prostate cancer (Hi-MYC) where neural progenitor cells are labeled yellow (DCX-eYFP), these cells are found throughout the prostate, similar to DCX expression in human tumors.    Additionally, these prostate DCX+ cells express markers of neural progenitors (e.g., nestin, CD24) without markers of stem cells (e.g., SOX2). (Credit: Mauffrey et al., 2019)

Figure 2: In a mouse model of prostate cancer (Hi-MYC) where neural progenitor cells are labeled yellow (DCX-eYFP), these cells are found throughout the prostate, similar to DCX expression in human tumors. Additionally, these prostate DCX+ cells express markers of neural progenitors (e.g., nestin, CD24) without markers of stem cells (e.g., SOX2). (Credit: Mauffrey et al., 2019)

A small gripe I have with the above figure has to do with the statistics used. For panel 2b, the authors state the data were analyzed using a one-sided Student’s t-test, which is a test that should be used for simple comparisons between two groups when there is strong evidence to suggest the outcome you are looking for is true. In biology, one-sided tests are rarely used, and the use of one here suggests that the authors were rather liberal when assigning significance…even though to the eye, it seems like the data would be significant with a two-tailed test, or a 2-way ANOVA with a more conservative post-hoc test (e.g., Tukey’s HSD).

Sorry for that tangent…moving back onto the paper, the authors provided evidence that DCX+ neural progenitors in the tumor differ in a few different ways from progenitors found in the brain. Specifically, they did not express markers of stem cells (e.g., SOX2) or markers of activated neural stem cells (e.g., GFAP, GLAST, CD133…). Instead, they expressed markers of neural progenitors (e.g., nestin, CD24). This can be seen in Fig. 2d, where samples from the brain (OB, SVZ) are compared to prostate tumor samples at 16 weeks or 52 weeks following cancer-induction. Strangely, in the text the authors describe that these cells showed neuron-differentiation and neuron-projection signatures, and say these data can be found in Figure 2fbut this panel does not exist.

Figure 3: Neural progenitors in the prostate differentiate into adrenergic neurons during tumor development. (   Credit:       Mauffrey et al., 2019)

Figure 3: Neural progenitors in the prostate differentiate into adrenergic neurons during tumor development. (Credit: Mauffrey et al., 2019)

After showing that DCX+ neural progenitors are present in their mouse model of prostate cancer (Hi-MYC), they went on to see if these cells ‘commit’ to a lineage and become a certain type of neuron. Specifically, they asked whether the cells would differentiate into neurons that produce the neurotransmitter norepinephrine (noradrenaline), which are called ‘adrenergic neurons’. This is important because adrenergic neurons can have wide-ranging effects on tumor growth, cancer progression, and metastasis. In the nervous system, DCX+ cells that migrate to the olfactory bulb (OB) usually ‘commit’ to the interneuron cell fate, allowing them to integrate into the circuits that process smell information. In Fig. 3, we can see that when cells were extracted from the brain (OB) or when cells were collected from the prostate tumor and grown in a dish (in vitro), they were able to differentiate into mature neurons (NF-H+), suggesting that these cells can commit to a terminal neural fate. Specifically, when looking in the organism (in vivo), Lin- eYFP+ neural progenitors were present in the tumor (blue color in Fig. 3 f,g,h), they fluctuated in amount during the course of tumor growth, and sent projections (axons) throughout the tumor tissue (Fig. 3 d,e). When co-labeled with an antibody against tyrosine hydroxylase (TH; a key enzyme in the norepinephrine synthesis pathway), they observed that DCX-eYFP+ cells also expressed TH, suggesting that they are indeed adrenergic neurons.

Figure 4: Neural progenitors in the brain (SVZ) migrate through the blood towards the prostate tumor in the Hi-MYC mouse model of pancreatic cancer.    Note: red (TdTomato) cells that originated in the brain’s SVZ could be found in the tumor throughout tumor development! Credit: Mauffrey et al., 2019).

Figure 4: Neural progenitors in the brain (SVZ) migrate through the blood towards the prostate tumor in the Hi-MYC mouse model of pancreatic cancer. Note: red (TdTomato) cells that originated in the brain’s SVZ could be found in the tumor throughout tumor development! Credit: Mauffrey et al., 2019).

So now we know that there are neural progenitors that colonize the tumor and can differentiate into adrenergic neurons (TH+). The question then becomes…where do these cells come from and how do they know how to get all the way to the tumor? They started by looking in the brain at different neural progenitor cells, and how their numbers change over time. They noted that a sub-population (green in Fig. 4) of Lin-eYFP+ progenitors changed in the SVZ during tumor growth, adding that this may be evidence of some of these cells leaving the area or the brain altogether (to putatively migrate to the tumor). To test this, they injected a lentiviral vector encoding the fluorescent protein TdTomato (red) into the SVZ to track where the cells go (as cells coming from that region will be labeled red no matter where they go in the body). The showed that these tagged neural progenitor cells could be found in the prostate tumor environment by 8, 12, and 16 weeks following tumor induction, providing evidence that they did indeed make the migration out of the brain (Fig. 4 f,g,h)! Additionally, by labeling the vasculature (CD31+) around the SVZ, they showed that in mice with tumors, the blood brain barrier (BBB) was disrupted, suggesting that the neural progenitors are able to leave the brain because the BBB is not functioning as usual.

One note I want to make is that the lentivirus approach that they used is not ‘cell-type specific’, and cells besides their target population (DCX-eYFP+) were definitely labeled. Additionally, since the lentivirus can infect any cells in the area, and it can actually travel in the blood stream and label cells outside the brain…this represents a potentially significant caveat.

Figure 5: DCX+ progenitor cells regulate tumor development in mice. Mice lacking DCX+ cells grew tumors that were much less aggressive and invasive. (   Credit: Mauffrey et al., 2019).

Figure 5: DCX+ progenitor cells regulate tumor development in mice. Mice lacking DCX+ cells grew tumors that were much less aggressive and invasive. (Credit: Mauffrey et al., 2019).

Finally, a major unanswered question was the ‘so-what?’ question, that is, do these cells actually do anything in the tumor micro-environment, or are they just sitting there as bystander cells. To test this, the authors used another transgenic strategy to express the diphtheria toxin receptor (DTR) on DCX+ progenitor cells. This allowed them to specifically eliminate DCX+ cells, letting them test whether they do indeed influence how prostate cancer develops.

They further used a new cancer model (PC3-Luc), where tumor cells are implanted in a recipient mouse. These cells were additionally engineered to express firefly luciferase (Luc). This allows researchers to ‘see’ where the tumor cells are in each mouse non-invasively, simply by injecting luciferin and measuring the light that is given off using a specialized camera (measured in photons). Using these approaches, they demonstrated that tumors in mice lacking neural progenitor cells (DCX+ ablated) caused fewer lesions and prevented the engraftment of transplanted tumor tissue. This suggests that DCX+ progenitor cells are critical for the early stages of tumor development! More striking is the finding that selectively eliminating DCX+ cells in the SVZ significantly inhibited tumor development, adding credence to the idea that these cells really do migrate from the brain to the prostate to elicit their effects. In the opposite experiment (where they transplanted DCX+ cells into mice with established tumors), they observed enhanced tumor growth (Fig. 5 d,e)!

Together, this study has a few problems that may detract from it’s primary finding. However, if additional research demonstrates that this phenomenon is real, it could be a huge game changer for both neuroscience and cancer! If depleting neural progenitors becomes a viable option for tumor suppression, this would be a completely new avenue for the treatment of prostate cancer, and potential other malignancies as well! One huge question that remains to be answered is “what is the signal from the tumor that causes progenitor cells to migrate?”. Figuring out the answer to this will be of paramount importance in developing tangible and realistic therapeutics.

Sorry for the super long post, but I just got really into this paper! Leave a comment below and join the discussion! For the latest updates, as always, follow me on twitter @jborniger. ‘Till next time, stay curious!