By Randall C Willis

 

Yet again, the patient returns to the oncologist’s office, braced to hear that her cancer has returned and hoping that it hasn’t spread.

 

What started as fatigue and a bit of stiffness has slowly evolved into years of biopsies, surgeries and chemotherapy. Time and again, she has thought she beat this thing that grows within her and yet just as regularly, it returns.

 

It’s frustrating to her, to her family. And she can see how frustrating it is even to her oncologist.

Just as they persevere in their battle to kill the tumor, so too does something within the tumor fight to stay alive.

 

Like the Hydra of Greek mythology who grew two new heads every time one was cut off, many tumor types follow a similar trajectory—they never completely succumb to the variety of surgical, radiotherapeutic, chemical and biological assaults we fling at them. Remove one set of cancerous cells and another set slowly take their place. Hit a tumor in one location and see it disseminate to others.

 

About 25 years ago, John Dick, now at Toronto’s Princess Margaret Hospital, speculated on the identity of this seemingly invulnerable core. Working on acute myeloid leukemia (AML), Dick transplanted human AML cells into SCID mice and noted their migration to the bone marrow niche, where they proliferated and reproduced disease similar to that in the human patient.

 

Recognizing that most human AML cells have limited proliferative capacity, Dick and colleagues wondered if the disease was maintained by a small population of cells with stem-like characteristics. Indeed, a limited dilution series showed that one cell in 250,000 from peripheral blood of AML patients had leukemia-initiating capacity.

 

Further characterization showed that these leukemia-initiating cells expressed the same surface markers—CD34⁺/CD38^-—as human adult stem cells. The concept of leukemia stem cells, and cancer stem cells (CSCs) more broadly, was born. And in the intervening years, CSCs have been discovered in a wide variety of tumors, both solid and liquid.

 

“This subset of cells is endowed with the ability to self-renew and differentiate into non-CSCs, indicating their capability of reproducing the tumor of origin when transplanted into immunocompromised mice,” wrote Giorgio Stassi and colleagues at University of Palermo in a recent review. “CSCs are also considered responsible for the metastatic spreading and chemoresistance. Strong evidence suggests that conventional treatments, including radio- and chemotherapy, spare the CSC subset, which is responsible for minimal residual disease (MRD) and cancer relapse.”

 

They explained that in part, that invulnerability to radiotherapy and chemotherapy is due to the metabolic differences between CSCs, including increased expression of drug transporters, enhanced activity of DNA-damage repair pathways and maintenance of a quiescent state compared to other rapidly dividing tumor cells.

 

“These features, combined with the capability of CSCs to evade the immune system, to activate an epithelial to mesenchymal transition (EMT) program and to adapt their metabolism under scarce nutrient conditions, render CSCs almost an imperishable cancer population,” the authors continued.

 

Thus, the theory goes, while chemotherapy may destroy the bulk of a tumor, it effectively enriches for CSCs and may even activate CSCs to grow and differentiate.

 

In fact, according to Stassi and colleagues, chemotherapy-induced damage stimulates CSCs in glioblastoma multiforme and bladder cancer to divide, repopulating the bulk tumor.

 

Another factor playing a role in CSC biology is the tumor microenvironment (TME), which acts through both intrinsic and extrinsic means, according to Fudan University’s Lun-Xiu Qin and colleagues.

 

“The intrinsic mechanisms include DNA methylation or demethylation and gene mutation, while the extrinsic actions involve the production of diverse growth factors and cytokines by the TME, leading to the activation of specific signaling pathways,” the researchers explained last year.

 

The importance of the TME, they suggested, was highlighted in a study showing that CSCs co-injected into mice with TME stromal cells formed much more aggressive tumors than CSCs injected alone.

 

“The [CSC] niche can contain various cell types and growth factors providing a tumor-promoting microenvironment,” the authors pressed. “This can involve endothelial cells, immune cells, cancer-associated fibroblasts (CAFs), various growth factors, and cytokines. In addition to these components, environment changes, such as hypoxia and pH have been proposed to contribute to the CSC niche.”

 

Hypoxia may be particularly important, they continued, as it helps maintain the stemness and thereby the malignancy of the cells. This also factors into the preference of CSCs toward glycolytic metabolism and a shift from OXPHOS. (For more on this, see the June 2019 DDN Special Report titled “Metabolic renaissance?”)

 

Not everyone is convinced, however, that there is a dedicated subpopulation of CSCs within each tumor.

 

“What I believe is that when tumors develop in different tissues, they retain in their developmental stages some aspects of the normal differentiation of that tissue,” states University of Manchester’s Peter Stern. “It’s a dynamic situation.”

 

“What tumors retain is some capacity to recapitulate the sort of stem-cell niche either after they’ve spread or even within the primary tumor itself,” he continues. “There’s some sort of stemness, if you like, but basically this also means the ability to divide and not differentiate.”

 

For Stern and many others, the concept of CSCs is more about the dynamic capacity of at least some tumor cells to flip between a more primitive, stem-like state and a more terminally differentiated state.

 

Tumors that are more differentiated, Stern continues, can be more tractable to treatments like radiation and chemotherapy. However, although cancers can show differences in their degree of differentiation, additional genetic changes are accrued during the natural history of any tumor through the failure to correct DNA replication errors, which leads to significant heterogeneity that can provide advantages if selected by natural or exogenous therapeutic challenge.

 

In many tumors, it appears that retaining a small population of cells able to find a niche that takes those cells out of the cell cycle, reinstating their stemness and making them more resistant to radiation and drugs that target the cell division machinery aren’t going to work.

 

Whatever the origin story for CSCs, being able to target them for therapeutic intervention requires further characterization of their unique biology and a search for markers that might allow us to not only identify them within an excess of other tumor cells, but also potentially use these markers to drive therapies.

 

Although CSCs have been isolated on the basis of cell surface markers such as CD133, CD44 and EpCAM, these markers are rarely unique to CSCs. There are many examples of different research groups isolating CSCs from the same types of cancers using completely different surface markers, yet examples of co-expression of these markers within a CSC population are rare.

 

“Another widely used method for obtaining ‘CSCs’ is to enrich them with suspension culture in defined CSC medium without serum,” offered University of Maryland’s Xiaoming He and colleagues earlier this year. “Although hanging drops, gyratory rotation and spinner flask, and NASA rotary cell culture systems have been developed to enrich CSCs via suspension culture, ultralow attachment plates (ULAPs) are most commonly used to enrich CSCs in suspension for various types of cancers.”

 

Such methods rely on the ability of CSCs to survive suspension culture due to their relative resistance to anoikis, apoptosis induced when cells detach from the surrounding extracellular matrix. As He and colleagues discovered with human breast, prostate and ovarian cancers, however, the theory doesn’t always hold up in practice.

 

“We cultured the four types of cancer cells at 1–20,000 cells per well in the ULAP,” the authors recounted. “Surprisingly, we noticed that more than 90 percent of the cells could survive and grow into aggregates or spheroids under the suspension culture.”

 

“This suggests that the suspension culture method for CSC isolation based on the anoikis of non-stem cancer cells is questionable as CSCs are considered to be rare in the cancer cell population,” they continued.

 

To surmount this challenge, the researchers took inspiration from embryonic development to engineer a microfluidic system that captured individual cancer cells within nanoliter-scale hydrogel microcapsules.

 

“Since recent studies show that the 3D hydrogel/scaffold may induce anoikis of non-stem cancer cells, we applied an alginate-based hydrogel scaffold in the core of the microcapsules,” the researchers explained. “Alginate is used because it is highly biocompatible and does not have cell adhesion molecules. The latter is good for inducing anoikis of non-stem cancer cells.”

 

When cultured, the single cells within these microspheres (1csc culture) were able to form tighter aggregates than those arising from ULAP culturing, and their self-renewal/stemness capacity was confirmed using a serial passaging assay.

 

RNA-Seq and RT-PCR experiments further showed that these colonies exhibited gene expression patterns consistent with CSCs, including altered expression of pathways related to cell structure organization, cell proliferation and stress-related responses. 1csc cells were also capable of differentiation into endothelial, cardiac, osteogenic and neural cell lineages.

 

Having confirmed cell stemness in vitro, the researchers then examined the ability of the cells to induce tumors in vivo, injecting them into immunodeficient mice. They found that tumors produced by the 1csc cells grew faster and larger than cancer cells grown by other methods. The cells were also capable of metastasis in another mouse line.

 

“We next treated these cells with two chemotherapeutic drugs, doxorubicin hydrochloride and camptothecin-11,” the authors continued, finding that the 1csc colony cells were significantly more resistant to both drugs than the cells produced by the other culture methods.

 

Taking a different tack, Perlmutter Cancer Center’s Theodore Welling and colleagues focused their CSC isolation efforts on the understanding that to induce metastasis, CSCs must leave their niche in the primary tumor and move through the bloodstream as circulating tumor cells.

 

Rather than rely on CTC isolation methods that use surface markers like EpCAM, the researchers turned to a device that utilizes size-based differential CTC focusing, testing their device with peripheral blood from patients with hepatocellular carcinoma (HCC).

 

Using several surface markers associated with HCC, the researchers were able to identify circulating tumor cells (CTCs) from both HCC cell lines and patient blood samples, and noted that the number of CTCs was slightly higher in patients with later-stage HCC.

 

“To examine whether HCC CTCs had a cancer stemness phenotype, three-color [immunofluorescent] staining was performed for all subjects using an additional anti-CD44 antibody,” the authors explained, finding that CD44+ CTCs represented the major proportion of CTCs across all stages of HCC.

 

Furthermore, the researchers detected CTC clusters called circulating tumor microemboli (CTMs) in more than half of the patients, offering further insight into tumor progression.

 

“Our approach can be readily applied in future clinical studies by incorporating single-cell genomic and transcriptomic profiling of CTCs isolated from the Labyrinth device,” the team suggested. “This will provide opportunities to further study the mechanisms by which CTCs and CTMs contribute to the process of invasion, metastasis, and recurrence.”

 

A significant challenge of characterizing CSCs in isolation, however, is that they don’t biologically function in isolation, but as described earlier, interact with the TME and other cellular partners. Thus, despite best efforts like those described above, there will always be questions about interpretation of data from such experiments.

 

“The best analogy I can give you is Heisenberg’s uncertainty principle,” says Stern. “You can’t measure the position and the momentum of a particle at the same time. In order to measure where it is, you have to put energy into the system, and that changes it.”

 

The same, he suggests, is true for CSCs.

 

“I think that it’s extremely hard to work out how, if you take something out, it will ever have the same properties that it might have had if you’d left it where it was,” he offers.

 

That said, CSC isolation methods have facilitated the identification and characterization of an ever-widening array of surface markers that researchers hope to target with new therapeutics.

 

The question, however, is whether this represents more noise than signal.

 

“The idea that you’re going to identify a marker that identifies all cancer stem cells is not very likely,” says Stern.

 

In fact, rather than focus on any given marker as a road sign for a tumor, Stern prefers to consider the underlying biological rationale for that marker.

 

The more important question for Stern is why that marker is expressed, and in his case, the marker of interest is the 5T4 oncofetal glycoprotein.

 

“It was originally defined as something that is expressed by human trophoblasts,” he notes. “And we were looking for shared properties of trophoblasts and cancer stem cells.”

 

Just as trophoblasts of the placenta function to protect the fetus within a genotypically distinct organism—Mom—so too does the tumor require protection from its host.

 

“The sort of thing we were looking for was something that might influence invasion and be associated with differentiation,” Stern continues.

 

Studies of embryonic stem cells and their differentiation pointed to 5T4, the presence of which also correlated with the risk of relapse in pediatric acute lymphoblastic leukemia (ALL).

 

“So, it’s an embryonic antigen and it’s shared by cancer cells,” says Stern.

 

Recently, Stern and colleagues Richard Harrop of Oxford BioMedica and Eric O’Neill of University of Oxford reviewed the current understanding of 5T4, particularly its role in CSC mobilization and potential therapeutic targeting.

 

“Overexpression of the 5T4 gene in different cell types is characterized by morphological changes, inhibition of cell–cell interaction, E-cadherin down-regulation, cytoskeletal disruption, reduced adherence and increased motility,” the authors described.

 

Key to the concept of cancer metastasis may be the influence of 5T4 on the regulation of chemokine and Wnt pathways.

 

Stern is quick to note, however, that he wouldn’t call 5T4 a CSC marker.

 

“I would say that it’s a marker of a property that might enable cancer stem cells to mobilize and also be expressed on some of the more differentiated tumor cells, which makes it quite an attractive target by comparison for delivering therapy where it would be specifically targeted at those cells,” he reframes.

 

Stern and colleagues have attempted a number of therapeutic approaches targeting 5T4, seeing mixed results.

 

Despite promising results from Phase 1 and 2 clinical trials, for example, modified vaccinia Ankara-5T4 vaccine TroVax (developed with Oxford BioMedica) did not meet its primary endpoint in a Phase 3 study in metastatic renal cell carcinoma, failing to show survival benefits in the overall patient population. That said, survival was improved in patients able to mount an immune response, suggesting that the vaccine may yet be worth evaluating for a subset of patients.

 

More recently, Stern and colleagues have focused their attention on the antibody-drug conjugate candidate A1mcMMAF, which links an anti-5T4 humanized monoclonal antibody to the microtubule-disrupting agent monomethyl auristatin F (MMAF).

 

In a 2017 report, University of Manchester’s Owen McGinn, Stern and colleagues initially tested the ADC in mice engrafted with an ALL cell line, and noted both reduced tumor growth and prolonged survival from A1mcMMAF versus control ADC or untreated animals.

 

They then tested the ADC and dexamethasone, as monotherapy or in combination, on patient-derived xenograft ALL mice. Although both monotherapies improved outcomes, the greatest survival benefits were seen with sequential administration of dexamethasone and A1mcMMAF.

 

Last year, University of Manchester’s Louise Wan, Stern and colleagues effectively repeated these experiments, but against ovarian cancer cell-engrafted mice and with a different chemotherapeutic partner.

 

“With A1mcMMAF treatment, there was an initial decrease in tumor load with the 4-day dosing regime,” the authors noted, adding that tumor burden increased following treatment with control ADC.

 

Before A1mcMMAF treatment was completed, however, tumor load began to rise back toward the original baseline.

 

“Tumors from the A1mcMMAF-treated animals continued to express 5T4 even when cytostasis was no longer maintained, suggesting that tumor escape is not caused by the outgrowth of 5T4-negative cells,” the authors related.

 

The researchers then tested A1mcMMAF and platinum-based chemotherapy carboplatin, both as monotherapies and in combination in xenograft mice.

 

“Treatment with carboplatin monotherapy…attenuated tumor growth rates but tumor load did not fall below baseline with these regimes,” the authors reported, whereas mice treated with A1mcMMAF monotherapy showed an initial decline in tumor load that was not sustained.

 

By comparison, combination therapy resulted in a continued reduction in tumor load, as well as a prolonged cytostatic effect after treatment cessation. And compared to treatment with A1mcMMAF alone, combination with carboplatin increased median survival by 71 percent.

 

The researchers discussed this synergistic effect.

 

“Conventional therapy tends to reduce tumor burden and improves symptoms but may fail to eradicate tumor-initiating cells, leading to eventual recurrence and drug resistance,” they suggested. “Therapies targeting tumor-initiating cells may, therefore, work best alongside conventional therapies that debulk the tumor mass.”

 

Such debulking steps may be particularly important for solid tumors, adds Stern, as therapeutics like antibodies are quite large and can be limited in their capacity to reach the target cells. Initially shrinking the tumors using chemotherapy might facilitate immunotherapeutic access.

 

NeoTX Therapeutics and Active Biotech, meanwhile, are working on immunotherapy with naptumomab, which fuses the Fab fragment of an antibody targeting 5T4 with an engineered bacterial superantigen that activates T cell responses.

 

In preclinical studies, naptumomab has shown synergistic anti-tumor effects and has extended overall survival when combined with checkpoint inhibition.

 

At the 2018 AACR meeting, researchers tested naptumomab and an anti-PD-1 as monotherapy or in combination in low immunogenic mouse tumor models. Although naptumomab alone showed some capacity to activate T cells and increase tumor infiltration and the anti-PD-1 had no effect, the combination more dramatically increased serum cytokines and CD8:CD4 ratio in the tumors, resulting in reduced tumor burden and prolonged media survival.

 

Findings like these led to a Phase 1b/2 clinical trial, currently recruiting, that combines naptumomab with AstraZeneca’s anti-PD-1 durvalumab versus advanced and metastatic solid tumors. The first patient in this study was dosed last October.

 

“The dosing of the first patient in our Phase 1b trial is a significant milestone for NeoTX, as it is our first clinical-stage molecule developed with our Selective T Cell Redirection (STR) platform,” said company CEO Asher Nathan in announcing the milestone. “Previous clinical studies have shown Nap to be well tolerated, and preclinical work conducted by the NeoTX team supports its broad potential in treating advanced and metastatic tumors, especially in combination with checkpoint inhibitors.”

 

In 2016, Robert Hawkins of the Christie CRC Research Centre and colleagues reported on a Phase 2/3 clinical trial of naptumomab plus interferon-α versus IFN-α monotherapy in renal cell carcinoma.

 

Although the addition of the fusion protein did not improve median overall (OS) or progression-free survival (PFS), thus missing the study’s primary endpoint, post-hoc analysis suggested that naptumomab showed synergistic effects in a subpopulation of patients.

 

Specifically, in subjects with sub-median levels of anti-naptumomab antibodies and IL-6 levels, median OS/PFS more than doubled from 31.1/5.8 months for IFN-α monotherapy to 63.3/13.7 months for the combination therapy.

 

As with so many other therapeutic efforts, one challenge to developing CSC-targeted immunotherapies may be the animal models on which they are tested. University of Milan’s Fabrizio Marcucci and colleagues elaborated on this challenge in their examination of ADC development versus CSCs.

 

“It is rather quite common that the compounds tested in animal models lead to complete tumor regressions, yet the same compounds fail in the human setting (i.e., the ADC rovalpituzumab tesirine),” the authors wrote. “In other terms, the predictability of animal models is very limited and it seems appropriate to say that observing complete tumor regressions in animal models is, nowadays, a necessary but by far not sufficient condition.”

 

Part of the challenge, they suggested, is because the percentage of injected dose taken up by the human tumor is much lower than in its mouse counterpart. With such a larger plasma volume in humans—liters vs. milliliters—much less of the dose/gram tumor tissue makes it into the tumor.

 

“Such a dose may be sufficient to eradicate a tumor in a mouse, but insufficient to eradicate an equivalent tumor in a human,” the authors explained. “Incidentally, it has been recently demonstrated that the intratumoral payload concentration correlates with the antitumor activity of ADCs, a result that underscores the importance of attaining a sufficient antibody or ADC concentration within a tumor tissue in order to be efficacious.”

 

They also questioned the ability of mouse tumor models, including PDXs, to faithfully recapitulate the heterogeneity found in the human setting, and they specifically pointed to stressors in the TME that may influence the conversion of non-CSCs to CSCs.

 

That said, the authors did not advise dropping the effort to create anti-CSC ADCs, but instead offered two different approaches to improve the likelihood of success.

 

One approach was to develop and improve in-silico models of human tumors to better predict the efficacy of antibodies or ADCs. Secondly, they called for increased efforts to identify biomarkers predictive of anti-tumor response, anti-CSC response or both.

 

“Elucidation of the relationship/lack of relationship between anti-CSC activity and clinical efficacy could allow to identify the contribution of anti-CSC activity to the overall antitumor activity and predict the efficacy of novel ADCs,” the researchers suggested.

 

In keeping with the targeted immunotherapy theme, CAR T cells are also being explored as potential modulators of CSCs.

 

As Rowa Alhabbab of King Abdulaziz University suggested in a review earlier this year, CAR T cell therapies present significant challenges but the possible rewards are high.

 

“Although CAR T cells are associated with several disadvantages such as their restricted efficiency, systemic immunogenicity, undesirable toxicity, and high cost, as well as the extensive time that is required for production, the huge success seen in their use with hematological malignancies and the continued investigations to overcome all these obstacles make CAR T cells a hugely promising therapy to treat cancers,” Alhabbab wrote. “Nevertheless, all immunotherapeutic approaches, including immune checkpoint inhibitors, are still at their initial steps of development and, therefore, are associated with challenges that have to be further studied and resolved, including Treg induction, toxicity, primary as well as acquired resistance, and limited efficiency.”

 

Earlier this year, Tim Leutkens of the Huntman Cancer Institute and colleagues described their efforts to develop a CAR T cell therapy targeting CD229 on multiple myeloma and tumor-propagating cells. As they noted, CD229 was an attractive target because of its strong and homogeneous expression on tumor cells as well as chemotherapy-resistant progenitors, and yet almost complete absence on non-cancerous cells.

 

The researchers first tested the CD229 CAR T cells against myeloma cell lines, as well as primary tumor cells from patients, and noted high cytotoxic activity in vitro. They then engrafted myeloma cell lines into mice and noted treatment resulted in significant loss of in-vivo imaging luminescence and prolonged survival.

 

Furthermore, they found that not only did treatment reduce the terminally differentiated cells, but also it reduced the number of tumor-propagating cells, as indicated through a colony-forming assay.

 

Taking the CAR T battle to solid tumors, Hongjiu Dai of Kaedi Biotech and colleagues recounted their efforts to tackle glioblastoma and its CSCs by targeting natural killer group 2 member D (NKG2D) ligands.

 

“The existence of cancer stem cells (CSCs) in glioblastoma is considered one of the major causes of the conventional therapy failure and cancer recurrence,” the authors noted. “Previous studies have shown that the targeted killing of CSCs could effectively inhibit glioblastoma tumorigenesis and prolong the survival of glioma-bearing mice.”

 

Another incentive to go the CAR T route was evidence suggesting the T cells were able to cross the blood-brain barrier, a challenge not easily overcome by many other therapeutics.

 

To verify the cytotoxic capabilities of the NKG2D-BBz CAR T cells, the researchers tested them in vitro against multiple glioblastoma cell lines and a control. Compared to CAR T cells with other specificities, the NKG2D-BBz CAR T cells efficiently lysed the glioblastoma cells but did not damage the control cells. And not surprisingly, the researchers observed that cells that expressed lower levels of NKG2D ligands were less sensitive to NKG2D-BBz CAR T cells.

 

The scientists then examined the activity of NKG2D-BBz CAR T cells against glioblastoma CSCs by generating suspended cell spheres from primary tumor. Flow cytometry highlighted that the cell spheres strongly expressed stem cell markers.

 

Again, NKG2D-BBz CAR T cells showed potent cytotoxicity and cytokine release when incubated with the suspended cell spheres, unlike the controls.

 

NKG2D-BBz CAR T cells also reduced and reversed tumor growth in subcutaneous xenograft of glioblastoma cell lines in mice. Furthermore, the authors wrote, “the results showed that NKG2D-BBz CAR-T cells significantly decreased the percentage of NESTIN-positive cells in tumors.”

 

This data paralleled the in-vitro findings and suggested that treatment also had cytotoxic effects on glioblastoma stem cells in vivo.

 

In discussing their findings, Dai and colleagues suggested that because NKG2D-BBz CAR T cells can lyse immunosuppressive cells, treatment may be able to eliminate the immunosuppression caused by the TME. As well, previous CAR T work has shown that full-length murine NKG2D could be detected in brain tissue eight months after inoculation, suggesting that NKG2D-BBz CAR T cells might survive longer than other CAR T cells in brain tumors.

 

And importantly, they noted, because NKG2D-BBz CAR T cells target multiple tumor-associated ligands, treatment could potentially avoid cancer immune escape often associated with tumor heterogeneity.

 

Despite these early successes, the use of CAR T cells as a monotherapy versus solid tumors has met with very limited success so far in clinical trials, according to Alhabbab.

 

“Therefore, one of the suggested strategies to increase the efficiency of CAR T cell therapy is to combine it with other therapeutic regimes such as chemotherapy and radiotherapy,” the author suggested.

 

“Several studies have reported that combining CAR T cells with chemotherapy can reduce the disease-associated side effects, improve the recognition of the tumor antigens, and enhance CAR T cell efficiency and persistence,” Alhabbab explained. “This enhanced efficiency was also seen upon combining CAR T cell therapy with radiotherapy.”

 

Perhaps we should not find it surprising that to reach the heart of a multi-headed, rapidly morphing beast—whether the Hydra or cancer—might require us to wield a multiplicity of armaments. For now, the search for better weaponry goes on.