This poster was originally written by Charlotte R Bell, Tania Katopodi, Yin Xin Ho, Emily Wright, Amy Cantrell, Nick Moore, Stewart Brown, Paul Farrington, Lorraine Mooney and Jane Kendrew Translational Oncology, Sygnature Discovery. It was presented at ELRIG Drug Discovery 2023.
Introduction
- Radiation therapy is a primary treatment modality for many types of cancer: approximately 50% of all cancer patients receive radiation therapy as part of their treatment regimen1.2
- Tumor irradiation (IR) can induce direct cytotoxic effects on cancer cells and modulate the tumor immune microenvironment (TIME)3.4
- Therapies that augment anticancer immune responses, such as immune checkpoint inhibitors (ICIs) targeting the PD-(L)1 axis, have revolutionized cancer treatment in recent years.5
- Several clinical trials are evaluating combinations of radiotherapy + ICI3
- Characterization of the effects of IR + ICI on TIME and tumor growth is therefore valuable to inform preclinical drug discovery research.
Methods
- MC38 colorectal carcinoma cells were implanted subcutaneously into female C57BL/6 mice and tumor growth was monitored 3 times per week using a caliper.
- Animals were randomized into treatment groups once tumors reached approximately 0.1 cm3
- Targeted X-rays were administered using an Xstrahl CIX3 irradiator. Animals were shielded by lead panels, with only the tumor exposed for localized IR treatment.
- Anti-PD-1 (RMP1-14) and anti-PD-L1 (10F.9G2) ICIs were purchased from BioXCell and administered intraperitoneally (IP) twice weekly at a dose of 10 mg/kg
- Tumors analyzed by flow cytometry were excised, digested into a single cell suspension, and stained with the antibody panel below. Samples were acquired on an Attune NxT flow cytometer (ThermoFisher Scientific)
Source: ELRIG (UK) Ltd.
| Channel | Marker pen | Fluorochrome |
|---|---|---|
| VL-1 | PD-1 | BV421 |
| VL-2 | alive/dead | efluor506 |
| VL-4 | CD4 | BV711 |
| BL-1 | CD8 | FITC |
| YL-1 | CD11b | Physical education |
| YL-2 | CD62L | PE-CF594 |
| YL-3 | CD45 | PE-Cy5.5 |
| YL-4 | FOXP3* | PE-Cy7 |
| RL-1 | F4/80 | Alexa Fluor 647 |
| RL-2 | CD3 | Alexa Fluor 700 |
| RL-3 | CD44 | APC-efluor 780 |
*intracellular antibodies
1. Fractionated IR results in tumor growth inhibition in the MC38 tumor model
Figure 1. Fractionated IR is effective and well tolerated in mice bearing C57BL/6 MC38 tumors. (a) Study design, (b) mean tumor volume (± SEM), (c) individual tumor volume, (d) individual % change in body weight over 1 week of treatment. n = 9 or 10 per group, CR = complete response. Image credit: ELRIG (UK) Ltd.
2. IR + ICI combinations improve tumor growth control
Figure 2. Combinations of fractionated IR with ICI have improved efficacy compared to monotherapies. (a) Study design, (b) individual animal tumor volume over time, (c) percent change in tumor volume 2 weeks after treatment. n = 10 per group, *p<0.05, **p<0.01, ****p<0.0001 as determined by Kruskal-Wallis with Dunn's multiple comparisons test. Image credit: ELRIG (UK) Ltd.
3. IR + ICI combinations modulate intratumoral immune infiltration
Figure 3. Flow cytometry gating strategy on MC38 tumors. Image credit: ELRIG (UK) Ltd.
Figure 4. Changes in intratumoral immune composition induced by IR + ICI combinations at day 9 of treatment. (a) Frequency of lymphoid and myeloid CD45+ cells and tumor cells/CD45- as a percentage of living cells, (b) frequency of tumor-associated macrophages (TAMs) as a percentage of CD45 cells+ cells, (c) CD3 frequency+ T and CD8 cells+ T cells as a percentage of CD45+ cells, (d) PD-1 frequency+ cells in percentage of CD8+ T cells, (e) frequency of regulatory T cells (Tregs) as a percentage of CD3+ T cells. Bars represent mean ± SEM, n = 5 or 6 per group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 as determined by one-way ANOVA with Tukey's multiple comparisons test. Image credit: ELRIG (UK) Ltd.
Summary
- Fractionated radiotherapy and immune checkpoint inhibitor combinations show significantly improved efficacy in the MC38 tumor model
- Combination of fractionated radiotherapy with anti-PD-(L)1 immune checkpoint inhibitors impacts the tumor immune microenvironment
- These data demonstrate that the MC38 tumor model is a valuable tool for preclinical research on drug discovery in immuno-oncology.
The references
- Baskar R, Lee KA, Yeo R, Yeoh KW, 2012. Cancer and radiotherapy: current advances and future directions. Int. J. Med. Sci. 9(3), pp.193-199
- Public Health England, 2020. [Online]. Chemotherapy, radiotherapy and surgical resection of tumours in England. Available from: https://www.gov.uk/government/statistics/chemotherapy-radiotherapy-and-surgicaltumour-resections-in-england/chemotherapyradiotherapy-and-surgical-tumourresections-in-england (accessed 02.05.23)
- McLaughlin, M, Patin, EC, Pedersen, M, Wilkins, A, Dillon, MT, Melcher, AA, Harrington, KJ, 2020. Remodeling of the inflammatory microenvironment by tumor cells after radiotherapy. Nat. Rev. Cancer 20(4), pp.203–217
- Rodriguez-Ruiz, ME, Vitale, I, Harrington, KJ, Melero, I, Galluzzi, L, 2020. Immunological impact of radiation-induced cell death signaling on the tumor microenvironment. Nat. Immunol. 21(2), pp.120–134
- Sharma P, Goswami S, Raychaudhuri D, Siddiqui BA, Singh P, Nagarajan A et al., 2023. Immune checkpoint therapy – current perspectives and future directions. Cell 186(8), pp.1652-1669
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