Targeting TGFβ

Nearly half of colon cancer patients—40 to 50 percent—face metastasis of their disease, according to a recent IRB press release, with secondary tumors appearing most often in the liver and lungs. Trying to combat this metastasis is difficult since, as the authors note in their paper, “no prevalent mutations have been associated with metastatic colorectal cancers. Instead, particular features of the tumor microenvironment, such as lack of T-cell infiltration, low type 1 T-helper cell (TH1) activity and reduced immune cytotoxicity or increased TGFβ levels predict adverse outcomes in patients with colorectal cancer.”

While prevailing mutations might be scarce, the authors reported that there are some hallmarks of this disease, as “Progression of colorectal cancer (CRC) generally coincides with successive alterations in four signalling pathways: WNT, EGFR, p53 and TGFβ. Mice bearing compound mutations in these four pathways were recently shown to enable the study of CRC metastasis. We crossed mice bearing conditional alleles in homologues of four key human CRC mutations: Apcfl/fl, KrasLSL-G12D, Tgfbr2fl/fl and Trp53fl/fl (designated A, K, T and P, respectively), and targeted gene recombination to intestinal stem cells (ISCs) by means of the Lgr5eGFP-creERT2 driver, which we designated L.”

The IRB team leveraged AMSBIO’s proprietary 3D organoid technology to develop a mouse model capable of simulating advanced colon cancer in humans. AMSBIO’s offerings feature a variety of matrices and solutions for 3D cell culture, including natural hydrogels, recombinant matrices, artificial scaffolds and scaffold-free systems, solutions that can support 3D cell culturing research.

“The ability to culture and maintain tumor organoids has been key to the success of this research,” said ICREA researcher Eduard Batlle of IRB. “Organoids, as provided by AMSBIO, offer multiple advantages as preclinical models for cancer research and drug testing.”

This was not an easy process by any means, however. Daniele Tauriello, postdoctoral fellow and first author of the article, noted that “The development of the animal model took us four years, but we hit the nail on the head.”

Once the team had their animal model, they confirmed the similarity of the resulting mouse tumors with human tumors. Armed with that confirmation, they generated a biobank of tumor organoids that were then grafted into immunocompetent mice. The new mouse model replicates the primary characteristics of metastatic colon cancer, providing a way to monitor how elevated levels of TGF-beta allow cancer cells to evade immune detection.

What they found with this mouse model was that, per the study, “Inhibition of the PD-1–PD-L1 immune checkpoint provoked a limited response in this model system. By contrast, inhibition of TGFβ unleashed a potent and enduring cytotoxic T-cell response against tumor cells that prevented metastasis. In mice with progressive liver metastatic disease, blockade of TGFβ signalling rendered tumors susceptible to anti-PD-1–PD-L1 therapy.”

When the researchers performed cell-population profiling of either mouse or human colorectal cancer samples, they found that cancer-associated fibroblasts “were the main contributors of TGFβ production.” Treating the mice with galunisertib, a TGFBR1-specific inhibitor, beginning 11 days after the transplantation of mouse tumor organoids “reduced primary tumor size, reduced the extent of carcinomatosis and blocked the appearance of liver metastases. Immunohistochemical quantification showed that galunisertib reduced the number of pSMAD3+ cells, and gene expression profiling demonstrated decreased levels of TGFβ-response signatures in fibroblasts and T cells.”

The IRB team also looked at the effects of galunisertib treatment specifically on liver metastases, and noted a “robust immune response,” one they theorized might inoculate mice against tumor cells. When they challenged the mice with the same mouse organoid tumors, “most tumors were rejected within two weeks in the continued absence of treatment.”

While treatment with galunisertib in mice with overt metastatic disease decreased metastatic burden, it “resulted in few complete remissions,” though the authors did note “increased infiltration of CD4+ T cells and of T-bet+ lymphocytes immediately after initiation of therapy.”

The scientists expect this discovery to have a rather immediate impact on the treatment of colon cancer patients, noting that “Oncologists and pharmaceutical companies will soon start clinical assays that combine TGF-beta inhibitors, which are already in clinical use, with immunotherapies. We are convinced that many colon cancer patients will benefit from this therapeutic strategy.”