Aside from surgical resection, locally advanced rectal cancer is regularly treated with neoadjuvant chemoradiotherapy. Since the concept of cancer treatment has shifted from only focusing on tumor cells as drivers of disease progression towards a broader understanding including the dynamic tumor microenvironment (TME), the impact of radiotherapy on the TME and specifically the tumor immune microenvironment (TIME) is increasingly recognized. Both promoting as well as suppressing effects on anti-tumor immunity have been reported in response to rectal cancer (chemo-)radiotherapy and various targets for combination therapies are under investigation. A literature review was conducted searching the PubMed database for evidence regarding the pleiotropic effects of (chemo-)radiotherapy on the rectal cancer TIME, including alterations in cytokine levels, immune cell populations and activity as well as changes in immune checkpoint proteins. Radiotherapy can induce immune-stimulating and -suppressive alterations, potentially mediating radioresistance. The response is influenced by treatment modalities, including the dosage administered and the highly individual intrinsic pre-treatment immune status. Directly addressing the main immune cells of the TME, this review aims to highlight therapeutical implications since efficient rectal cancer treatment relies on personalized strategies combining conventional therapies with immune-modulating approaches, such as immune checkpoint inhibitors.

Treatment of patients with locally advanced rectal cancer (RC) is based on neoadjuvant chemoradiotherapy followed by surgery. In order to reduce the development of therapy resistance, it is necessary to further improve previous treatment approaches. Recent in vivo experimental studies suggested that the reduction of tumor hypoxia by tumor vessel normalization (TVN), through the inhibition of the glycolytic activator PFKFB3, could significantly improve tumor response to therapy. We have evaluated in vitro and in vivo the effects of the PFKFB3 inhibitor 2E-3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) on cell survival, clonogenicity, migration, invasion, and metabolism using colorectal cancer cells, patient-derived tumor organoid (PDO), and xenograft (PDX). 3PO treatment of colorectal cancer cells increased radiation-induced cell death and reduced cancer cell invasion. Moreover, gene set enrichment analysis shows that 3PO is able to alter the metabolic status of PDOs toward oxidative phosphorylation. Additionally, in vivo neoadjuvant treatment with 3PO induced TVN, alleviated tumor hypoxia, and increased tumor necrosis. Our results support PFKFB3 inhibition as a possible future neoadjuvant addition for patients with RC. Significance: Novel therapies to better treat colorectal cancer are necessary to improve patient outcomes. Therefore, in this study, we evaluated the combination of a metabolic inhibitor (3PO) and standard radiotherapy in different experimental settings. We have observed that the addition of 3PO increased radiation effects, ultimately improving tumor cell response to therapy.

Supplementary Fig. 6 from Tumor Vessel Normalization via PFKFB3 Inhibition Alleviates Hypoxia and Increases Tumor Necrosis in Rectal Cancer upon Radiotherapy

Supplementary Fig. 2 from Tumor Vessel Normalization via PFKFB3 Inhibition Alleviates Hypoxia and Increases Tumor Necrosis in Rectal Cancer upon Radiotherapy

3PO potentializes irradiation effects in colorectal cancer cells. A, Colony formation assay performed with HCT-116, HT-29, SW-1463, and SW-837 cancer cells upon 3PO treatment. Cells treated with DMSO or 2.5 µmol/L 3PO for 4 hours and exposed to indicated doses of RT. Six days (HCT-116), 7 days (HT-29), and 14 days (SW-1463 and SW-837) later, colonies were stained and counted. Data are displayed as means ± SD, n = 3. ∗, P < 0.05; one-way ANOVA. B, Colony formation assays performed with HCT-116, HT-29, SW-1463, and SW-837 cells treated with different concentrations of 3PO for 4 hours, exposed to either RT (6 Gy) or not irradiated and analyzed after 6 days (HCT-116), 7 days (HT-29), 14 days (SW-1463), and 14 days (SW-837) by counting colonies. C, Invasion assay performed with HCT-116, HT-29, SW-1463, and SW-837 cancer cells. Cells were plated in a Boyden multi-well chamber and treated with 10 µmol/L 3PO for 4 hours, subjected to RT (6Gy), and incubated for a period of 96 hours. Data are displayed as means SD, n = 3. ∗∗, P < 0.01; ∗∗∗∗, P < 0.0001; one-way ANOVA. D–F Cell migration assay quantification of HCT-116, HT-29, and SW-837 cancer cells upon combinatory treatment. Cells were treated with 3PO for 4 hours and subjected to RT (6 Gy). Data are displayed as means ± SD, n = 3. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001; one-way ANOVA.