Evaluation of molecular-level changes of Programmed cell Death Ligand-1 after  radiotherapy in a BALB/c CT26 colorectal mouse tumor model

doi:10.21203/rs.3.rs-1620965/v1

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Evaluation of molecular-level changes of Programmed cell Death Ligand-1 after radiotherapy in a BALB/c CT26 colorectal mouse tumor model

https://doi.org/10.21203/rs.3.rs-1620965/v1

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The effects of radiation therapy (RT) for cancer can be systemic and partially mediated by the immune system. However, radiation alone is unlikely to transform an immunosuppressive environment into an immunostimulatory one. Therefore, an effective combination of radiation therapy and immunotherapy may provide a new more efficient treatment approach. Here we investigated how the expression of programmed cell death-ligand 1 (PD-L1) in the microenvironment of the tumor varied in different RT regimens with the same Biologically Effective Dose (BED). In this study, female BALB/c mice inoculated with CT26 tumor cells were irradiated with three different RT regimens using the same Biologically Effective Dose (BED) of 40 Gray (Gy). These included Ablative RT (1*15 Gy), Hypo-fractionated RT (2*10 Gy), and Conventional RT (10*3 Gy). PD-L1 expression was analyzed with immunohistochemical staining on days 2, 20, and when the size of tumors had reached 2 cm² after RT. All treated groups expressed PD-L1, but the group receiving single ablative high dose RT showed higher expression compared to the other groups. No significant differences in PD-L1 expression were observed at different times in the same group. These findings showed that different regimens of RT have different effects on the tumor microenvironment (TME), so a combination of RT and immune checkpoint blockade could be clinically used in cancer patients.

Radiation therapy

biologically effective dose

tumor microenvironment

programmed cell death-ligand 1

immunohistochemistry

mouse tumor model

Radiation therapy (RT) is one of the major therapeutic modalities used in the treatment of locally advanced solid tumors and is used to treat about 50% of all cancer patients. Also, 40% of patients who showed improvements continue to use it to manage their disease. For some time research into improving the results of radiation therapy has focused on the cancer cell itself, regardless of the complex interaction that occurs between the cancer cell and the tumor microenvironment (TME). Over the last two decades, radiotherapists have moved towards considering the effects of the TME. Radiation therapy works in a variety of ways, including direct destruction of tumor cells, modulation of the TME, and stimulation of the host immune response against the tumor cells (1–3). RT can exert a wide range of cytotoxic effects against tumors, which at the molecular level cause non-repairable DNA strand breaks that ultimately lead to cellular senescence and apoptosis. Moreover, RT causes a specific type of cell death called immunogenic cell death (ICD), which is characterized by the release of danger signals or DAMPs (damage associated molecular patterns). DAMPs that occur due to ICD after RT include cell surface exposure of calreticulin (CRT), the extracellular release of ATP and high mobility group box 1 (HMGB1) (4).

Since RT can have both stimulatory and inhibitory effects on the immune system, it should be combined with immunotherapy (IT) to activate the immune system and overcome any immunosuppressive effects it may have. Some preclinical and clinical studies have been performed using this combination, and some good results have been obtained. It is clear that TME components are not only involved in tumor progression, but also in the response to treatment (5), so a better understanding of changes in TME following RT will help us to design improved therapeutic combinations. Programmed death-ligand 1 (PD-L1) is one of the molecules that inhibit the activity of immune cells within the TME, and upon interaction with programmed death 1 (PD-1) receptor expressed on cytotoxic T cells, it plays an essential role in decreasing their attack against the tumor cells. So PD-L1 seems to be a good target for combining immunotherapy with other anti-cancer treatments such as RT (10, 11). Mimura et al. showed that PD-L1 expression was positively correlated with the presence of CTLs in the stroma, and IFN-γ expression in the tumor, and that expression of PD-L1 was a key limiting factor for CTL activity (8).

In our previous study (9), when an anti-PD-L1 antibody was combined with high single-dose radiation therapy we observed an increase in IFN-γ and tumor-infiltrating CTLs in the tumor, resulting in increased antitumor activity. As a result, the combination therapy resulted in effective tumor control, increased survival, and finally, no recurrence of the tumor was observed. Dovedi et al. showed that tumor cell expression of PD-L1 was increased after fractionated RT (10). Studies have shown that combining RT with immune checkpoint inhibitors promotes localized tumor control and the release of tumor antigens that activate the immune system (11). In addition, to improving the treatment results, the optimal dose of radiation in each session should be considered (12, 13). Accordingly, in radiobiology, the concept of Biologically Effective Dose (BED) based on a Linear Quadratic (LQ) model indicates the biological effect of RT treatment according to changes in the dose per fraction. Therefore, the BED describes the cell response to RT and is used to compare different treatment regimens (14).

Therefore, as a continuation of our previous study, and considering that PD-L1 expression is dependent on IFN-γ production, in this study we examined the expression of PD-L1 and IFN-γ after RT with various doses and fractionation in a BALB/c mouse CT26 tumor model. We sacrificed the mice at days 2, 20, and when the size of tumors reached 2cm² after RT. The difference in PD-L1 expression after radiotherapy regimens with the same BED allowed the optimal therapeutic dose to be determined.

Cell line and mouse model

For our study, we used the CT26 cell line, which is a mouse epithelial colon adenocarcinoma line. To culture CT26 cells, RPMI-1640 medium was used with 100 U/mL penicillin, 100 µg/mL streptomycin, 20% heat-inactivated fetal bovine serum (FBS) and 1% L-glutamine. All cells were maintained at 37 ° C in 5% CO2. These cells were also free from Mycoplasma infection.

All experiments on animals as well as their care were performed with the approval of the ethics committee of Shahid Beheshti University of Medical Sciences with the approval number: IR.SBMU.MSP.REC.1397.616. BALB/c mice 5–6 weeks old were purchased (Pasteur Institute of Iran, Tehran, Iran) and kept in standard conditions with 12 hour light/dark cycle and free access to food and water.

Tumor challenge

For in vivo injection, mice were anesthetized by intraperitoneal (IP) injection with ketamine (100 mg/kg) and xylazine (12.5 mg/kg). Then 1 × 10⁶ CT26 tumor cells were injected subcutaneously into the right flank of the anesthetized mice (15).

Treatment

The BED equivalent of 40 Gy was delivered with three different RT regimens, including ablative (15 Gy × 1), hypo-fractionated (10 Gy × 2) and hyper-fractionated (3 Gy × 10), the dose in each regimen was calculated based on LQ models.

14 days after tumor cell injection the mice were randomly divided into 3 treatment groups (each group consisting of at least 12 mice) plus 1 group as a control. 18 days after the initial inoculation, when the size of tumors reached at least 300–400 mm³, all groups except the control group started the irradiation. The Ablative RT regimen was a single fraction of 15 Gy on day 18, the hypo-fractionated RT regimen was 2 fractions of 10 Gy delivered on days 18 and 28, and the conventional (hyper-fractionated) RT regimen was 10 fractions of 3 Gy delivered daily from day 18 to day 31 as shown in Fig. 1.

The anesthetized mice were treated with radiation using a linear clinical accelerator (6 MV photons, Elekta synergy linear accelerator, Stockholm, SE), with ethical considerations taken to to minimize their suffering. Since only the tumor area should be irradiated, the mouse body except the irradiated field was protected by a 9 cm thick lead plate. RT was delivered to a 3×3 cm² field with 5-mm margins at 350 Gy/min with 6 MV X-rays using tangential beam delivery. A layer of Superflab Bolus Material of 1.5 cm was placed on the tumor and the radiation source was adjusted to a distance of 100 cm from the skin.

At the end of the treatment period, the mice were sacrificed at specific times, 2 days, 20 days, and finally when their tumor size reached 2cm², and their tumor tissues were isolated. The tissue was divided into two parts. One part of the tissue was prepared for flow cytometry analysis, while the remaining part was immediately fixed in formalin. After the fixation process, the tumor tissues were embedded into paraffin wax blocks.

Flow cytometry analysis

Isolated tumor tissue was digested with type I collagenase in RPMI-1640 and incubated at 37 ° C for 2 hours. The cell mixture was filtered through a 70 µm cell strainer and then centrifuged at 1,500 rpm for 10 min. These cells were stained by specific antibodies. Cells were suspended in flow cytometry staining buffer containing PBS + 5% FBS and analyzed by flow cytometry (BD FACS Calibur flow cytometer, Becton Dickinson, USA) using fluorochrome-labeled antibodies against PD-L1 (Biolegend, 155404), IFN-γ (clone XMG1.2), CD8 (clone 53–6.7) and IgG1 isotype control (clone RTK2071) (Biolegend, San Diego, California). The CD8 is a surface antigen, so fluorescent antibody was added to the cells followed by 30 min incubated at 4 ° C and finally washed twice with staining buffer. IFN-γ is an intracellular marker, so cells were fixed and permeablized by True-Nuclea^tm Transcription Factor Buffer Set (BioLegend) kit before adding the fluorescent antibody. After reading the samples by flow cytometry, the results were analyzed with FlowJo 7.6.1 software (16).

PD-L1 Immunohistochemical Analysis

Tumor sections (4 µm thick) were cut from formalin-fixed, paraffin embedded tumor blocks, and mounted on positively charged glass slides. For the deparaffinization step, tumor slides were placed twice in xylene for 10 minutes at room temperature. Subsequently, sections were immersed successively in absolute ethanol, 90% ethanol, 70% ethanol, 50% ethanol followed by one rinse with water. For antigen retrieval sections were incubated with Tris-EDTA buffer, pH 9.0 for 30 minutes at 95°C and then cooled down for 40 minutes. Endogenous peroxidase and proteins were blocked. Slides were incubated with primary antibody against PD-L1 (ab233482 anti-mouse IgG1) for 1 hour at a concentration of 10 µg/mL according to a standard protocol. After washing in TBS buffer, the secondary antibody (envision Dako, K4006) that was conjugated with HRP was applied for 30 minutes at room temperature. After each incubation step, the slides were washed with TBS buffer. Incubated in substrate solution for 5 minutes and washed with distilled water, then counterstained with hematoxylin for 1 minute. Finally dehydrated in graded ethanol, cleared in xylene, mounted on a coverslip and examined with a light micrscope. The staining results were interpreted by experienced pathologists who were blinded to the groups. Cancer cells that showed membranous and cytoplasmic staining for PD-L1 were considered positive cells. When tumor cell staining with any intensity was 1–5%, 5–10%, or 10–20% in each section containing approximately 100 tumor cells, the samples were classified 1+, 2+, and 3 + respectively.

Tonsil and placenta tissues were stained as negative and positive controls with and without primary antibody.

Statistical Analysis

Descriptive statistics were analyzed using the independent t-test, and the post hoc test for one-way ANOVA analysis of variance carried out by GraphPad Prism version 5 (GraphPad Software, San Diego, CA, USA).

Evaluation of PD-L1 expression in CT26 tumor cells in control group

At the first step, the tumor tissues of the control group of mice that did not receive RT were assessed for PD-L1 expression both by FACS and IHC. After tumor induction in mice, the tumors were isolated and examined for expression of PD-L1. PD-L1 expression was confirmed by the median fluorescence intensity (MFI) vale in the FACS histogram (Figure 2A) and also by IHC staining of sections (Figure 2B).

IFN-γ expression in the ablative RT group showed the highest MFI of IFN-γ compared to the other RT groups

The histogram of IFN-γ expression showed an increased MFI in the ablative RT group, with a 4.43 fold increase in IFN-γ expression in immune cells within the tumor tissue compared to unstained control. In the other two regimens there was a comparative decrease in IFN-γ expression with a difference of P>0.05 between regimens with the same BED (Figure 3).

CD8+ IFN-γ+ cells were higher in ablative treatment group than in the other groups

Because of the increase of IFN-γ after treatment, CD8+ IFN-γ+ double-positive cells in the tumor were examined. The Ablative RT regimen increased the infiltration of CD8+ cells expressing IFN-γ more than that the hypo-fractionated and conventional regimen groups (Figure 4).

Ablative RT showed higher PD-L1 expression compared to other RT regimens with the same BED

After confirming the expression of PD-L1 on the surface of CT26 tumor cells, RT treated tumor tissues were examined for this marker. The results of the analysis showed that the CT26 tumor tissues of mice who received the ablative RT regimen had 3+ expression with 10 to 20% of cells showing cytoplasmic and membranous staining, while in the hypo-fractionated group, 5 to 10% of tumor cells showed membranous and cytoplasmic staining with a score of 2+, and in the conventional RT group only 1 to 5% of tumor cells showed staining with a score of 1+ (Figure 5).

Moreover, we analyzed and compared the expression of PD-L1 in tumor tissues sampled at different times, 2 days after treatment, 20 days after treatment, and when the size of the tumors had reached 2cm². We observed that there were no significant differences between the different time points.

Overall, we showed that the high expression of PD-L1 after ablative RT treatment could be associated with high expression of IFN-γ in this group. The results of our study showed that the expression of IFN-γ in the ablative group has the highest MFI while in the immunohistochemical analysis we observed that the expression of PD-L1 in the ablative group also had the highest expression.

Considering that no study has examined PD-L1 expression after different RT regimens so far, in this study we measured the expression of PD-L1 after three different RT regimens with the same BED, so that we could make a rational decision on an appropriate combination therapy. Our study, like other studies in this field, confirmed that RT can affect the immune system and alter the microenvironment of the tumor.

Also, we showed that each type of treatment regimen induces different effects, although the BED of these three treatment regimens was the same. Our findings showed significantly greater expression of PD-L1 after ablative RT than hypo-fractionated and conventional RT, but there was no significant difference between the expression of PD-L1 at different times, which could be confirmed by increasing the number of samples. These findings are consistent with previous studies that PD-L1 expression is correlated with IFN-γ production, and is important for immunotherapy based on blockade of the PD-1/PD-L1 axis (17, 18). Dovedi et al. also showed that the treatment of tumor cells with a range of RT doses in vitro did not have a direct effect on PD-L1 expression, and further suggested that CD8 + cells produce the IFN-γ, and are responsible for up-regulating PD-L1 expression in tumor cells following RT (10).

On the other hand, several studies have shown that RT can induce activation of anti-inflammatory pathways. For instance, the secretion of the immunosuppressive cytokine transforming growth factor-beta (TGF-β) was increased by RT with a single dose of 5 or 10 Gy (19). These are just a few examples of the effects of RT on the immune system and the TME, each of which affects the progression and response of tumors to treatment. This poses many questions relevant to tumor biology and cancer treatment.

Since PD-L1 expression is not limited to tumor cells alone, and is also expressed by other cells within the tumor, including DCs, natural killer (NK) cells and tumor associated neutrophils (TAN), it is necessary to examine PD-L1 in other cells. It is clear that PD-L1 is usually expressed on the tumor cells and inhibits the function of PD-1 expressing T cells. As a result, the tumor has the opportunity and ability to escape from attack by the immune system (20). Deng et al. showed that PD-L1 expression was increased on DCs and tumor cells after a single 12Gy dose of RT (21). Dong et al. found that NK cells could be divided into two groups based on the presence or absence of PD-L1, and suggested that PD-L1 positive NK cells had significant cytotoxicity and higher IFN-γ production compared to PD-L1 negative NK cells. They showed that an anti-PD-L1 antibody directly potentiated the anti-cancer effect of NK cells, independently of the expression of PD-L1 on the surface of the tumor cells. In this regard, the anti-PD-L1 monoclonal antibody can act directly on NK cells expressing PD-L1 to enhance the attack on the tumor, which may explain why anti-PD-L1 has the same therapeutic effect on tumors without any PD-L1 expression on the tumor cells. (22).

Furthermore, Wang et al. suggested that TANs present in gastric cancer tumors could directly suppress the T cell immune response by the increased PD-L1 expression induced by GM-CSF (23). In addition to PD-L1, RT can also affect CTLA-4 expression. Katalin Balázs et al. suggested that RT (median of 57.6 Gy delivered over a median of 44 days) induced a strong increase in CTLA-4 levels on CD4 + T cells measured in cancer patients (24). Therefore, in future studies, the effects of various radiotherapy regimens on the expression levels of CTLA-4 should be considered, as another important inhibitory checkpoint in the TME.

Several previous studies in animal models, as well as the result of our present study have shown a link between increased PD-L1 expression and the increased production of IFN-γ. However, to date there has been only limited information in humans. The question of how exactly PD-L1 expression is increased at the tumor cell surface and in the TME after RT has not been clearly answered. On the other hand, it is known IFN-γ can have a dual effect on the anti-tumor immune response, because it can induce the expression of both PD-L1 and HLA class I at the same time. Therefore the anti-tumor activity of CTL could depend on the balance between the inhibitory effect of PD-L1, and the stimulatory effects of HLA-I, both induced by IFN-γ (8). Finally, the results of our study could help to select a suitable immunotherapy intervention (such as anti-PD-L1) to be combined with a single high dose of RT. However, more extensive studies are needed to examine the effects of RT on other cells and molecules involved in the anti-tumor immune response, in the TME.

Acknowledgment

This article has been extracted from the thesis written by Ms. Fateme Khani Chamani in School of Medicine, Shahid Beheshti University of Medical Sciences.

Author contribution

SAJ and MA designed the experiments; FKH performed experiments and collected data; AM, SAJ, MSH, GHMCH and MRH discussed the results and strategy; SAJ Supervised, directed and managed the study; FKH, MSH, AM, MA, GHMCH, MRH and SAJ Final approved of the version to be published.

Funding

This work has been supported by Shahid Beheshti university of Medical Sciences research ward by the grant number: 18162.

MRH was supported by US NIH Grants R01AI050875 and R21AI121700.

Data availability

The data of this research is accessible up on demand from corresponding author.

Competing interest: The authors have no relevant financial or non-financial interests to disclose.

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Evaluation of molecular-level changes of Programmed cell Death Ligand-1 after radiotherapy in a BALB/c CT26 colorectal mouse tumor model

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Introduction

Materials And Methods

Statistical Analysis

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