Chemoradiation has been used as the standard treatment plan for cancer patients to this day. However, the drug resistance developed by patients greatly limits the effectiveness of treatment. More and more studies have found that the occurrence of drug resistance is not only related to the resistance mechanism of tumor cells themselves, but also inextricably linked to changes in the tumor microenvironment.
It has been found that neutrophil infiltration provides a metastasis microenvironment for the planting of tumor cells, increasing the risk of liver metastasis of pancreatic cancer[1]; tumor cells expressing the oncogenic protein TRIB3 can inhibit CD8+ T cell infiltration, etc. [2]. Another "leader" in the tumor microenvironment is fibroblasts, which have a variety of functions, including promoting tumor development [3] and mediating tolerance to CAR-T therapy [4].
However, it is not known whether fibroblasts are involved in tumor tolerance to chemoradiotherapy.
Recently, the team of Professor Florian R. Greten of the German Cancer Research Center published a blockbuster study in the journal Cancer Cell [5] that explained that inflammatory tumor-associated fibroblasts (iCAF) are associated with chemoradiotherapy tolerance, cancer recurrence and shortened survival of patients.
Specifically, the large amount of IL-1a released by tumor cells after radiation therapy can not only polarize tumor-associated fibroblasts (CAF) into iCAF, but also cause oxidative DNA damage-mediated cellular senescence. These effects eventually lead to resistance to chemoradiation in patients, leading to disease progression.
It was also based on this finding that they proposed a treatment regimen of chemoradiation combined with IL-1 receptor blockers.
Screenshot of the first page of the paper
Let's take a look at how this study is conducted.
First, Greten's team found that the normal Unified Molecular Subtype Classification (CMS) approach to rectal cancer was not successful in predicting disease-free survival (DFS).
To explore molecules or signaling pathways that could have predictive potential, they used mass spectrometry to test the proteome of tumor cells in 61 patient samples. However, principal component analysis (PCA) of the results did not distinguish between patients with a good prognosis and a poor prognosis, suggesting that it is not the tumor cells themselves that may determine the prognosis of patients with rectal cancer.
To find the key factors that determine the prognosis of patients with rectal cancer, Greten's team conducted multicolor fluorescent immunohistochemistry experiments. The results showed that when comparing the various cell populations of patient samples with good and poor prognosis, only mesenchymal cells differed between the two groups. Further gene enrichment analysis (GSEA) also found high enrichment of iCAF-related genes in tumor samples from patients with poor prognosis.
The multicolor fluorescence test showed a difference in the proportion of mesenchymal cells in tumor tissue between the two groups with different prognosis
To further validate the function of iCAF in it as well as the underlying mechanisms, Greten's team built a mouse model of in situ organoid tumors.
On the original APTK (mutations in genes Apc, Trp53, Tgfbr2, K-rasG12D, etc.), they added myristicylated AKT and obtained the APTKA tumor organoid model. Previous studies have demonstrated that APTKA tumor models have the characteristics of interstitial-associated gene enrichment, greater invasiveness, and easier spontaneous liver metastasis [6].
Greten's team found that APTKA tumors after radiation therapy showed more liver metastases, accompanied by DCN+ fibroblasts, F4/80+ macrophages, and Ly6G+ neutrophilia, as well as CD8+ T cell reduction.
In APTKA in situ organoid mouse models, radiation therapy caused greater liver metastases with DCN+ fibroblasts
Curiously, if the APTK and APTKA tumor organoids were radiotherapy in vitro and then transplanted under the skin of mice, both types of tumor growth were inhibited, suggesting that the APTKA organoids' tolerance to radiation therapy was largely mediated by stromal cells.
To further verify whether APTK and APTKA organoid models can affect fibroblasts by paracrine, Greten's team treated primary fibroblasts of the small intestine with supernatants cultured with APTK and APTKA, respectively, RNA sequencing of fibroblasts after 24 h, and combined with the single-cell sequencing results of CAFs in APTKA mouse tumors, it was found that the IL-1-dependent cell cluster in APTKA tumors was the most important.
Later, Greten's team further found that compared with the APTK-derived supernatant, APTKA supernatant treatment can significantly activate the NF-κB and p38 signaling pathways, and the use of inhibitors of IKKβ and p38 can inhibit the expression of pro-inflammatory related genes.
Inhibitors of both IKKβ and p38 inhibit the expression of pro-inflammatory factors activated by APTKA supernatant
So which cytokine is this change related to in the APTKA supernatant?
To find this key cytokine, Greten's team sequenced RNA from APTK and APTKA organoids, respectively, and the analysis showed that there were large differences in the expression of some cytokines between the two types of organs.
They then used neutralizing antibodies corresponding to these cytokines to neutralize these cytokines and detected the expression of pro-inflammatory-related genes after APTKA supernatant treatment of fibroblasts. It was found that the pro-inflammatory-related genes expressed by the ATPKA supernatant activated by fibroblasts were suppressed only if IL-1a was neutralized. Further illustrate that the differentiation of iCAF is mediated by IL-1a.
So far, Greten's team has discovered that IL-1a is an important signal for tumor cells to transmit information with fibroblasts. Does that mean blocking the pathway reduces tolerance to treatment?
To test this idea, they treated mice with anakira, a blocker of IL-1 receptors, and then administered radiation therapy ten days later, and found that anakira was able to significantly reduce tumor burden and aggressiveness.
In vivo experiments in mice have also demonstrated that anakira can inhibit tumor aggressiveness
After that, Greten's team further analyzed how CAFs mediated the occurrence of resistance to radiation therapy. They found that after radiation treatment, APTKA supernatant-induced polarized fibroblasts produced p53-dependent senescence accompanied by changes in cyclin expression.
So can the above phenomena observed in mouse models be used to predict how rectal cancer patients will respond to treatment?
Greten's team collected serum samples from patients prior to treatment and found that IL-1a concentrations were below the detectable range, and that neither IL-1β nor most cytokines could predict a patient's prognosis. Interestingly, however, the concentration of IL-1ra in serum decreased significantly in patients with poor prognosis. Because IL-1ra is an antagonist molecule of the IL-1 signaling pathway, this phenomenon also indicates the presence of activation of IL-1 signaling in patients with poor prognosis.
Serum IL-1ra concentrations in patients with poor prognosis were lower than those with a good prognosis
This study shows that IL-1ra is directly linked to fibroblasts that cause poor prognosis in patients, and further provides evidence for the significance of CAFs in therapeutic sensitivity, as well as an important theoretical basis for future chemotherapy regimens in cancer patients.
Schematic of iCAF-induced radiotherapy and chemotherapy tolerance in patients with rectal cancer
bibliography:
1.Bellomo, G. et al. Chemotherapy-induced infiltration of neutrophils promotes pancreatic cancer metastasis via Gas6/AXL signalling axis. Gut gutjnl-2021-325272 (2022) doi:10.1136/GUTJNL-2021-325272.
2.Shang, S. et al. TRIB3 reduces CD8+ T cell infiltration and induces immune evasion by repressing the STAT1-CXCL10 axis in colorectal cancer. Science translational medicine 14, eabf0992 (2022).
3.Murray, E. R. et al. Disruption of pancreatic stellate cell myofibroblast phenotype promotes pancreatic tumor invasion. Cell reports 38, 110227 (2022).
4.Sakemura, R. et al. Targeting Cancer-Associated Fibroblasts in the Bone Marrow Prevents Resistance to CART-Cell Therapy in Multiple Myeloma. Blood (2022) doi:10.1182/BLOOD.2021012811.
5.Nicolas, A. M. et al. Inflammatory fibroblasts mediate resistance to neoadjuvant therapy in rectal cancer. Cancer Cell 40, 168-184.e13 (2022).
6.Varga, J., Nicolas, A., Petrocelli, V., … M. P.-J. of E. & 2020, undefined. AKT-dependent NOTCH3 activation drives tumor progression in a model of mesenchymal colorectal cancer. rupress.org.
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