Translating the Cross-Talk Between Tumor Microenvironment and Cancer Stem Cells

“I only know one thing: that I know nothing.” Attributed to Socrates, the quote embodies the complexity of knowledge and the importance of humility in its pursuit.

Nowhere is this truer than in cancer research. Each advancement in our understanding of tumor cells seems only to reveal the vast complexity and unknown mechanisms yet to be discovered.

Tumor cells have the ability to alter the surrounding tissue environment to support their own growth, differentiation, and self-renewal. They are engaged in constant, dynamic communication with their tumor microenvironment. The implications of these abilities are profound, multifaceted, and deeply interwoven with the body’s natural systems.

The multi-omics technologies developed over recent decades are unveiling the depth of this complexity, providing us with powerful tools to explore and decode these intricate biological systems, opening up new avenues for therapeutic interventions and precision medicine.

The first observed interaction between tumors and their microenvironment dates back to 1863.

However, it was only in 2000 that the seminal paper “Hallmarks of Cancer” proposed that cancer cells manifest through six essential alterations in cell physiology that collectively drive malignant growth – independent growth signaling, resistance to growth-inhibitory signals, evasion of apoptosis, uncontrolled replication, sustained angiogenesis, and tissue invasion.

These physiological changes represent a fundamental shift in cellular behavior that is not only determined by genetic mutations within cancer cells, but also by their ability to manipulate surrounding tissues to create conditions favorable for their survival and proliferation.

Cancer cells share their ability to self-renew and differentiate with the stem cells.

A pioneering paper published in 2001 drew parallels between tumor cells and stem cells. Both stem and cancer cells can self-renew and differentiate into heterogeneous tissue cells, suggesting that the pathways that regulate stem cells may also regulate cancer cells.

The concept of cancer stem cells (CSCs) and their interplay with the tumor microenvironment (TME) are today well established: the tumor microenvironment provides a supportive niche for CSC survival, self-renewal and maintenance of stemness properties, while the CSCs in turn create favorable conditions for progression, therapy resistance, and metastasis.

Scientists’ quest to decipher this complex cross-talk pushes the boundaries of knowledge. As technology advances, it unlocks new possibilities and allows researchers to find answers to questions previously beyond reach.

RNA-seq, commercially launched in 2007, has been critical in revealing the breadth of heterogeneity of the tumor and its interactions with the microenvironment. This method, however, has limitations in exploring the genetic heterogeneity of cancer tissue, as it averages transcription profiles across all analyzed cells.

High resolution technologies like single cell RNA sequencing (scRNA-seq) address the limitations of bulk RNA-seq by enabling whole transcriptome sequencing within individual cells, providing insights into the role of each cell within the ecosystem, uncovering rare cell types, and helping understanding cell differentiation processes and responses to various stimuli.

Cancer Stem Cells, a rare tumor cell subset, are key to tumor growth and treatment resistance. They self-renew, differentiate into various cancer cells, and can resist chemotherapy by entering a quiescent state. This adaptability allows CSCs to survive treatments that eliminate most cancer cells, potentially causing relapse. Effective therapies must target both CSCs and differentiated cancer cells.

CSCs’ self-renewal relies on pathways like Hedgehog, Notch, Wnt/beta-catenin, PI3K/AKT, and NF-kB. These pathways, normally regulating development and cell fate, become aberrantly activated in cancer due to genetic mutations or the tumor microenvironment, leading to uncontrolled proliferation, invasion, metastasis, and treatment resistance.

scRNA-seq has proven ideal for studying these rare cancer stem cells and their dysregulated pathways. A landmark 2014 study by Patel et. al applied scRNA-seq to five glioblastomas, revealing a complex cancer ecosystem with diverse transcriptional programs, including markers for stemness, differentiation, proliferation, and quiescence. The researchers identified meta-signatures related to cell cycle, hypoxia, immune response, and oligodendrocyte function.

Heterogeneous expression of receptor tyrosine kinases (RTKs) and signaling molecules, such as NOTCH2 and JAG1, has significant implications for glioblastoma treatment, as aberrant RTK expression may drive resistance to targeted therapies and immunotherapies. The stemness gradient and presence of quiescent stem-like cells suggest that targeting CSCs is crucial for effective treatment.

An Australian team extended this concept by using scRNA-seq with combinatorial barcoding to study druggable targets in Sonic Hedgehog (SHH) medulloblastoma, a rare, aggressive cancer of the cerebellum driven by abnormal SHH pathway activation. Researchers targeted the transcription factor OLIG2, crucial in SHH medulloblastoma, where OLIG2+ glial progenitors drive tumorigenesis and are enriched in therapy-resistant, recurrent tumors.

They used CT-179, a small molecule inhibitor that blocks OLIG2 function, slowing tumor growth and enhancing radiation and targeted therapies to prevent recurrence. In medulloblastoma, OLIG2-expressing CSCs survive conventional therapy, driving relapse. Using scRNA-seq and combinatorial barcoding, the team observed transcriptional changes post-treatment, noting G2/M cell cycle arrest without harming healthy oligodendrocytes. They also found upregulated CDK4 in resistant cells, suggesting CDK4 as a potential target for combination therapy.

Targeting OLIG2-expressing tumor stem cells may reduce recurrence and improve outcomes in medulloblastoma.

CSCs don’t exist in isolation. The tumor microenvironment, a complex ecosystem of immune cells, stromal cells, ECM, and blood vessels, supports CSC survival and self-renewal through reciprocal communication. CSCs, in turn, promote an immunosuppressive state in the microenvironment, challenging therapies that rely on the immune system.

Such a state represents a real challenge for cancer therapies harnessing the power of the immune system.

Tumor Infiltrating Lymphocytes (TILs), particularly CD4 and CD8 T cells, are key players in immunotherapy, offering potent anti-tumor activity. They cluster within the tumor microenvironment, often correlating with better prognosis and treatment outcomes. However, the tumor microenvironment can suppress immune responses by releasing inhibitory factors, leading to inconsistent TIL therapy effectiveness.

An MD Anderson study used scRNA-seq and TCR-sequencing to examine TILs in pancreatic ductal adenocarcinoma (PDAC), identifying 13 distinct TIL states, including cytotoxic CD8-GZMK and CD8-ZNF683 subpopulations crucial for anti-tumor responses. This research highlights how TILs transition between states in the tumor microenvironment, offering potential biomarkers for prognosis or immunotherapy responses.

This paper from Harvard took it further, profiling neoadjuvant-treated and treatment-naive PDACs samples using spatial molecular imaging, single-nuclei RNA-sequencing (snRNA-seq), and digital spatial profiling data. They mapped specific T-cell states and expansion patterns within pancreatic cancer tumors, providing insights that could help in developing more effective immunotherapies for PDAC. This work lays the foundation for future research and development of immunotherapies tailored to the specific immune landscape of pancreatic cancer.

The field of cancer research is rapidly evolving, driven by the complexity of the disease and the advancement of cutting-edge technologies. ScRNA-seq has emerged as a powerful tool, addressing many critical questions and providing deeper insights into the intricate and dynamic nature of cancer biology.

Despite these advancements, cancer remains a formidable challenge due to its ability to evolve and develop resistance to treatments. Addressing key questions — such as the identification of CSCs, the complexity of TMEs, and the mechanisms of drug resistance — requires continued technological innovation, multidisciplinary collaboration, and a commitment to personalized medicine.