A Ludwig Cancer Research study using human tissue samples and mouse models has discovered that recurrent tumors of the aggressive brain cancer glioblastoma multiforme (GBM) grow out of the fibrous scars of malignant predecessors destroyed by interventions such as radiotherapy, surgery, and immunotherapy. Led by Ludwig Lausanne’s Johanna Joyce, PhD, Spencer Watson, PhD, and alumnus Anoek Zomer, PhD, the researchers applied multi-omics analyses to understand how these scars enable the regrowth of tumors, and also identified drug targets to sabotage their malignant support. The team demonstrated the efficacy of a combination therapy in preclinical mouse models of GBM.

“We’ve identified fibrotic scarring as a key source of GBM resurgence following therapy, showing how it creates a protective niche for regrowth of the tumor,” said Joyce. “Our findings suggest that blocking the process of scarring in the brain by adding anti-fibrosis agents to current treatment strategies could help prevent glioblastoma from recurring and improve the outcomes of therapy.” Joyce and colleagues reported on their studies in Cancer Cell, in a paper titled “Fibrotic response to anti-CSF-1R therapy potentiates glioblastoma recurrence.”

GBM is the most common and aggressive form of brain cancer in adults. Despite considerable effort to develop effective therapies for the cancer the average life expectancy of patients remains around 14 months following diagnosis. “Standard-of-care (SoC) treatment for these high-grade gliomas includes surgery, temozolomide-based chemotherapy, and fractionated ionizing radiation (IR),” the authors wrote. However, they stated, “Nearly all glioblastomas eventually recur following treatment, underscoring the need to better understand therapeutic resistance mechanisms.”

The origins of the newly reported study date back to 2016, when the Joyce lab reported its examination in mouse models of strategies to overcome resistance to a promising immunotherapy for the treatment of GBM. That experimental therapy inhibits signaling by the colony stimulating factor-1 receptor (CSF-1R) and is being evaluated in clinical trials today. The approach targets macrophage immune cells and their brain-resident versions, microglia, both of which are manipulated by GBM cells to support tumor growth and survival.

The Joyce lab in addition demonstrated that CSF-1R inhibition reprograms these immune cells into an anti-tumor state and so induces significant tumor regression. Yet the team’s prior research in mouse models also indicated that about half the mice show relapse following an initial response to the therapy. “What was most remarkable about that observation was that every single time a brain tumor recurred following immunotherapy, it regrew right next to a scar that had formed at the original site of a tumor,” said Joyce. Referring in the Cancer Cell paper to their previous study, the team wrote, “Interestingly, histological analyses revealed that 100% of recurrent tumors regrew immediately adjacent to regions of glial scarring. By contrast, scars were only observed in 20% of the treated mice surviving to the trial endpoint, suggesting a potential mechanistic link between scarring and glioma recurrence.”

They also noted that despite recent insights into the role of fibrosis in primarily epithelial cancers and the correlations between extracellular matrix (ECM) gene expression and poor patient prognosis, “… it remains unclear how CNS scarring may impact the response to therapy in glioblastoma.”

For their new work Joyce, Watson, Zomer and their colleagues examined tumor samples obtained from patients undergoing GBM therapy and showed that fibrotic scarring occurs following therapy in humans as well—and that it is similarly associated with tumor recurrence. They also showed that the fibrotic scarring occurs in response to not only immunotherapy but also following the surgical and radiological removal of tumors. Immunofluorescence (IF) imaging of patient tissue identified “fibrotic responses immediately adjacent to the resection cavity, similar to our findings in mouse models of surgical resection,” they noted.

To explore how fibrosis contributes to GBM relapse the researchers applied an integrated suite of advanced omics technologies to analyze the cellular and molecular geography of the scars and the microenvironment of resurgent tumors.

These technologies include the analysis of global gene expression in individual cells, the comprehensive analysis of proteins in the tissues as well a workflow and AI-powered suite of analytical methods for the spatial analysis of tissues named hyperplexed immunofluorescence imaging (HIFI). Recently developed by Watson and colleagues in the Joyce lab, HIFI permits the simultaneous visualization of multiple molecular markers in and around cells across broad cross-sections of tissues, enabling the generation of granular maps of the tumor microenvironment.

“Applied together, these advanced methods allowed us to see exactly how fibrotic scars form,” said Watson. “They revealed that the fibrosis serves as a kind of protective cocoon for residual cancer cells and pushes them into a dormant state in which they are largely resistant to therapy. We found that it also shields them from surveillance and elimination by the immune system.”

Integrated analyses of the tumor microenvironment (TME) following therapy revealed that the descendants of cells associated with tumor-feeding blood vessels become functionally altered to resemble fibroblasts—fiber-producing cells commonly involved in wound-healing. These perivascular-derived fibroblast-like (PDFL) cells fan out across the region previously occupied by the regressing tumor, where they mediate the generation of fibrotic scars. “Our data also suggest that PDFLs are a key mediator of treatment-induced CNS scarring,” the investigators stated. These cells, the researchers found, are especially activated by neuroinflammation and immune factors known as cytokines, most notably one called transforming growth factor-β (TGF-β). “Collectively, our data indicate that anti-glioma therapies that aggressively perturb the glioblastoma TME, such as immunotherapy, radiotherapy, and surgery, have the potential to trigger a fibrotic treatment response,” they noted.

“To see if targeting fibrotic scarring could improve therapeutic outcomes for GBM we devised a treatment regimen using existing drugs to block TGF-β signaling and suppress neuroinflammation in combination with CSF-1R inhibition and evaluated it in preclinical trials using mouse models of GBM,” said Joyce. “We also timed these additional treatments to coincide with the period of maximal PDFL activation identified by our studies. Οur results show that the drug combination inhibited fibrotic scarring, diminished the numbers of surviving tumor cells and extended the survival of treated mice compared to controls.” The authors further noted, “|The fibrotic treatment response was mediated by perivascular-derived fibroblast-like cells via activation by transforming growth factor β (TGF-β) signaling and neuroinflammation. Concordantly, combinatorial inhibition of these pathways inhibited treatment-associated fibrosis, and significantly improved survival in preclinical trials of anti-colony-stimulating factor-1 receptor (CSF-1R) therapy.”

Noting limitations to their study, the researchers suggest that approaches to limit fibrotic scarring could significantly improve outcomes for GBM patients receiving surgical, radiation or macrophage-targeting therapies. Their findings, they stated, “… notably indicate that addressing treatment-associated fibrosis in the patient setting could improve the efficacy of multiple treatment modalities, including surgical resection and IR treatment, as these both stimulated fibrotic treatment responses.” Additional research, they suggested, will likely yield even better drug targets for such combination therapies.

Previous articleChemical Genetics Shows How “Undruggable” GTPases Can Be Targeted
Next articleSymbiosis Acquires New Bioprocess Facility in Stirling, Scotland