Radiosensitivity of Cancer Stem Cells Has Potential Predictive Value for Individual Responses to Radiotherapy in Locally Advanced Rectal Cancer

Abstract

Neo-adjuvant radiotherapy is frequently employed in the therapeutic management of locally advanced rectal cancer (LARC). Radiotherapy can both reduce local recurrence and improve the success of surgical procedures by reducing tumor mass size. However, some patients show a poor response to treatment, which results in primary resistance or relapse after apparent curative surgery. In this work, we report in vitro and in vivo models based on patient-derived cancer stem cells (CSCs); these models are able to predict individual responses to radiotherapy in LARC. CSCs isolated from colorectal cancer biopsies were subjected to in vitro irradiation with the same clinical protocol used for LARC patients. Animal models, generated by CSC xenotransplantation, were also obtained and treated with the same radiotherapy protocol. The results indicate that CSCs isolated from rectal cancer needle biopsies possess an intrinsic grade of sensitivity to treatment, which is also maintained in the animal model. Notably, the specific CSCs' in vitro and in vivo sensitivity values correspond to patients' responses to radiotherapy. This evidence suggests that an in vitro radiotherapy response predictivity assay could support clinical decisions for the management of LARC patients, thus avoiding radiation toxicity to resistant patients and reducing the treatment costs.

Keywords: cancer stem cells; in vitro radiotherapy; locally advanced rectal cancer (LARC); neo-adjuvant radiotherapy; preclinical model of radiotherapy.

Figure 1

Animal model-derived tumors presented the same phenotype as human parental cancer. (A) Hematoxylin and Eosin (H&E) staining, (B) CK20 and (C) CDX2 immunohistochemistry of the mouse xenograft. (D) Hematoxylin and Eosin staining, (E) CK20 and (F) CDX2 immunohistochemistry of human colorectal cancer biopsy. Arrows indicate the gland structures. Magnification 20×.

Figure 2

Cancer stem cells (CSCs) from different rectal biopsies show different in vitro sensitivity levels to radiotherapy. Proliferation modulation, in comparison with untreated cells, is reported for Line 1 (A), Line 2 (C), Line 3 (E) and Line 4 (G). Vitality modulation, in comparison with untreated cells, is reported for Line 1 (B), Line 2 (D), Line 3 (F) and Line 4 (H).

Figure 3

CSCs from different rectal biopsies showed in vivo sensitivity to radiotherapy comparable to that found under in vitro treatments. Graphical representations of in vivo tumor growth (%) measured at three different time points (0D, 15D, 30D) after treatment with 5 Gy dose radiation, supplied for 5 consecutive days, in combination with a dose rate of 2400 MU/min, in comparison with not treated sample (NT), for CSC Line 1 (A), Line 2 (B), Line 3 (E) and Line 4 (F). (C,D,G) Representative CT scan images taken the day of the last irradiation (D0) and 30 days after treatment for line 1, 2 and 3. Line 4 produce fast growing refractory tumors with consequent risk of ulceration. All mice implanted with line 4 had to be sacrificed at day 20 because they reached tumor mass limit. For this reason, CT scan images are not available. Representative images of ex vivo measures for line 4 (H).

Figure 4

In vitro treatment predicted the clinical outcomes of rectal cancer patients treated with neo-adjuvant radiation. Representative MRI images before (A) and after (B) the radiotherapy of a patient whose CSCs became sensitive to in vitro treatment. Representative MRI images before (C) and after (D) radiotherapy of a patient whose CSCs became resistant to in vitro treatment.