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Cancer Biomarkers -1 (2023) 1–15 1

DOI 10.3233/CBM-220268
IOS Press

A single targeted gamma-ray irradiation

induced an acute modulation of immune cells
and related cytokines in EMT6
mouse-bearing tumour model
Nurhaslina Hasana,b , Narimah Abdul Hamid Hasania , Effat Omara , Fatihah Ronny Shama ,
Syed Baharom Syed Ahmad Fuada , Muhammad Khalis Abdul Karimc and Mohammad Johari Ibahima,∗
a
Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh, Selangor, Malaysia
b
Faculty of Dentistry, University Teknologi MARA, Sungai Buloh, Selangor, Malaysia
c
Faculty of Science, Universiti Putra Malaysia, Selangor, Malaysia

Received 25 July 2022

Accepted 31 May 2023

Abstract.
BACKGROUND: A complicated interplay between radiation doses, tumour microenvironment (TME), and host immune system
is linked to the active participation of immune response.
OBJECTIVE: The effects of single targeted 2 Gy and 8 Gy gamma-ray irradiations on the immune cell population (lymphocytes,
B-cells, T-cells, neutrophils, eosinophils, and macrophages) in EMT6 mouse-bearing tumour models was investigated.
METHODS: The effects of both irradiation doses in early (96 hours) and acute phase (5 to 11 days) post-irradiation on immune
parameters were monitored in blood circulation and TME using flow cytometry. Simultaneously, selected cytokines related to
immune cells within the TME were measured using multiplex ELISA.
RESULTS: A temporary reduction in systemic total white blood count (TWBC) resulted from an early phase (96 hours) of
gamma-ray irradiation at 2 Gy and 8 Gy compared to sham control group. No difference was obtained in the acute phase.
Neutrophils dominated among other immune cells in TME in sham control group. Eosinophils in TME was significantly increased
after 8 Gy treatment in acute phase compared to sham control (p < 0.005). Furthermore, the increment of tumour necrosis
(TNF)-α, eotaxin and interleukin (IL)-7 (p < 0.05) in both treatment groups and phases were associated with anti-tumour activities
within TME by gamma-ray irradiation.
CONCLUSION: The temporary changes in immune cell populations within systemic circulation and TME induced by different
doses of gamma-ray irradiation correlated with suppression of several pro-tumorigenic cytokines in mouse-bearing EMT6 tumour
models.

Keywords: Mouse-bearing tumour model, gamma-ray irradiation, tumour microenvironment, cytokines

1. Introduction 1

The incidence of breast cancer among women has 2

∗ Correspondingauthor: Mohammad Johari Ibahim, Department increased tremendously in 2020 as compared to 2018, 3
of Biochemistry and Molecular Medicine, Faculty of Medicine, Uni-
surpassing the other common cancers such as colorectal 4
versiti Teknologi MARA Selangor Branch, Sungai Buloh Campus,
Jalan Hospital, 47000 Sungai Buloh, Selangor, Malaysia. Tel.: +60 and lung cancers [1,2]. Sadly, the actual number of cases 5

361267347/+60 167165065; E-mail: mji@uitm.edu.my. might be higher as data provided was based on pre- 6

ISSN 1574-0153/$35.00 c 2023 – IOS Press. All rights reserved.

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2 N. Hasan et al. / Immune cells and cytokines induced by gamma-ray

7 pandemic COVID-19 (coronavirus disease) era. During supplemented with 10% foetal bovine serum, 100 IU 54

8 the pandemic, the incidence was under-diagnosed, and m/L penicillin G, and 100 µg/mL streptomycin in 5% 55

9 delayed due to implementation of lockdown, reduced CO2 at 37◦ C until 70 to 80% confluent. Cells were sub- 56

10 availability of healthcare facilities, and suspension of cultured once every two to three days. Cells at 6th to 57

11 screening program. Subsequently, leading to increment 7th passages were used for the development of mouse- 58

12 in advanced stage diagnosis and poor survival rate [3]. bearing tumour model. All the chemicals and dispos- 59

13 Cancer treatments such as surgery and chemotherapy able equipments used were tissue culture grade (Gibco, 60

14 were frequently cancelled or postponed as these treat- USA). 61

15 ments potentially cause prolonged immunosuppression

16 and severe infection, especially to elderly patients who 2.2. Study design 62

17 were most vulnerable (Centers for Disease Control and

18 Prevention (CDC), 2021). Thus, the used of modified The experimental design was summarised in Fig. 1. 63

19 radiotherapy such as hypofractionation radiotherapy as A total of 36 female BALB/c mice aged between 6 64

20 alternative especially in the current situation was jus- to 8 weeks were divided into 6 groups. Group 1 con- 65

21 tified [4,5]. Here, higher dose of radiation given in a sisted of 6 healthy uninoculated and untreated mice 66

22 shorter duration of treatment was suggested for imme- which served as negative control. Group 2 or sham con- 67

23 diate tumour control management [4,6,7]. However, trol were inoculated with EMT6 cells, but not treated 68

24 more data were required as exposure to different higher with gamma-ray irradiation. Both groups 1 and 2 were 69

25 doses of radiation may caused chronic toxicity includ- euthanised at day 10th post-inoculation. Groups 3 to 70

26 ing erythema, epilation, ulceration, fibrosis and telang- 6 were inoculated and treated. In the early phase study, 71

27 iectasia [8]. Thus, more preclinical studies were needed groups 3 and 4 were euthanised at 96 hours (day 14th 72

28 to optimise benefits and minimise side effects due to post-inoculation) after receiving gamma-ray treatments 73

29 immunosuppression. at 2 Gy and 8 Gy, respectively. In the acute phase study, 74

30 The role of immune system in the regulation of tu- tumour growth in groups 5 and 6 were monitored within 75

31 mour control has been elucidated [9,10]. One of the 30 days post-irradiation. However, mice were sacrificed 76

32 interesting findings related to immune response within earlier if any one of these 3 factors were observed: (i) tu- 77

33 TME was a double-edge sword role of eosinophils in- mour size reached mortality mass, or volume reached > 78

34 filtration as pro- or anti-tumour agents with inconsis- 1000 mm3 or 11% of total body weight [23], (ii) more 79

35 tent results in different types of cancers [11–13]. The than 10% reduction in body weight or (iii) behavioural 80

36 effect of radiation-induced immune changes in tumour changes, such as aggressive and reduction of more than 81

37 control produced contradictory outcomes by either pro- 10% in food and water intake. All these criteria were 82

38 moting immunosuppression leading to poor tumour signs of tumour progression reducing quality of life. 83

39 ablation [14–18] or radiation related to immunostim- All procedures involving mice were conducted in ac- 84

40 ulant effect in enhancing recognition for cancer cell cordance with relevant guidelines and regulations by 85

41 death [19,20]. Active participation of immune reactivity USDA Pain and Distress Categories as approved by An- 86

42 either through bystander or abscopal effects, suggest imal Ethics Committee, Universiti Teknologi MARA 87

43 a complicated interplay between radiation, TME, and (Reference Number: UiTM Care 208/2017). The AR- 88

44 host immune system [21,22]. The capability of radi- RIVE guideline in minimizing the number of animal 89

45 ation in modulating TME is used as a platform in in- used, reducing pain suffered by these animal [24], and 90

46 vestigating the relationship between different doses of qualified personal in handling all the procedures were 91

47 single targeted gamma-ray irradiation with the implica- adhered. 92

48 tion of eosinophils infiltration, cytokine, and subsequent

49 impact on tumour growth. 2.3. Mouse-bearing tumour model 93

The detailed preparation of mouse-bearing tumour 94

50 2. Materials and methods model was previously published by [25]. Briefly, the 95

mice were acclimatised and bred in pathogen-free con- 96

51 2.1. Cell line ditions (Laboratory Control Unit, Faculty of Medicine, 97

Universiti Teknologi MARA). Prior to inoculation, 98

52 EMT6 mouse mammary carcinoma (ATCC, USA) the mice were anaesthetised with ketamine/xylazine at 99

53 was cultured in Dulbecco’s modified Eagle’s medium 0.1 mL/mg body weight. Subsequently, inoculation was 100
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N. Hasan et al. / Immune cells and cytokines induced by gamma-ray 3

Fig. 1. The experimental design for mice tumour inoculation, gamma-ray irradiation, and euthanisation during the early and acute post-irradiation
phases. Group 1: Mice healthy, uninoculated and untreated, Group 2: Mice inoculated but untreated, Groups 3 and 4: Mice inoculated and treated
with 2 Gy and 8 Gy gamma-ray, respectively (early phase), Groups 5 and 6: Mice inoculated and treated with 2 Gy and 8 Gy gamma-ray,
respectively (acute phase).

101 performed subcutaneously by injecting 10 µL of 1 x for flow cytometry and protein cytokine analysis. Sev- 130

102 105 EMT6 cells suspension into the right hind leg and eral mice died during experiment due to unforeseen 131

103 above the knee (stifle joint) of mice. Tumour growth circumstances were excluded from the study. The final 132

104 and body weight were monitored every 2 to 3 days surviving numbers for each Groups 1 to 6 were 6, 4, 6, 133

105 post-inoculation. The length and width of tumour was 5, 5 and 3, respectively. 134

106 measured twice using a digital calliper to calculate its

107 volume starting on day 5th post-inoculation until eu- 2.6. Blood analysis 135

108 thanisation Faustino-Rocha et al. [26].

Blood analysis was conducted using an automated 136

109 2.4. Gamma-cell irradiation blood analyser (XN-550, Sysmex Co., Kobe, Japan) at 137

the Centre of Pathology Diagnostics & Research Lab- 138

110 The tumour was irradiated using a Gamma Cell oratories, Universiti Teknologi MARA according to 139

111 220 unit (Nordion, Ottawa, Canada) at Faculty of Sci- the manufacturers protocol. Results were re-confirmed 140

112 ence and Technology, Universiti Kebangsaan Malaysia manually with white blood cell (WBC) differential 141

113 at an operating dose rate of approximately 18.67 count. 142

114 Gy/min. Groups 3 to 6 were irradiated on day 10th

115 post-inoculation, with either 28 Gy (Groups 3 and 5) 2.7. Flow cytometry analysis 143

116 or 75 Gy (Groups 4 and 6) delivery doses to achieve

117 respective 2 Gy and 8 Gy absorbed doses at targeted An approximate 0.2 mg tumour section was cut into 144

118 area. These irradiation steps were performed using our smaller pieces, crunched with mortar, and filtered using 145

119 custom-made lead shield and strainer [27]. The ab- a 70 µm cell strainer. After washing with phosphate 146

120 sorbed doses were chosen based on our previous work buffer saline (PBS), pellet was collected and resus- 147

121 by Ibahim et al. [25] and measured using a Gafchromic pended in a pre-warm lysis buffer to produce a single- 148

122 EBT3 film (Ashland Specialty Ingredient, USA). This cell suspension. 1 x 106 /mL cells were diluted in PBS 149

123 film was attached to the strainer and placed in the centre and incubated in the dark with fixable viability stain 150

124 of the chamber. (FVS) for 5 minutes at room temperature. After dis- 151

carding of supernatant and addition of blocking buffer, 152

125 2.5. Mouse euthanisation a cocktail of CD45, Siglec-F, IA-IE, CD 11b, and Ly6G 153

conjugated antibody (BD Biosciences PharmingenTM , 154

126 Prior to euthanisation, each mouse was anaesthetised California, USA) was added and incubated in the dark 155

127 and whole blood sample collected into EDTA tubes us- again for 30 minutes at room temperature. After wash- 156

128 ing cardiac puncture technique. Mice were sacrificed ing, cells were diluted in PBS and used for data ac- 157

129 by cervical dislocation. Tumours were excised and used quisition immediately. The samples were analysed by 158
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4 N. Hasan et al. / Immune cells and cytokines induced by gamma-ray

Fig. 2. The comparison of mean value for tumour growth volume between gamma-ray irradiated groups compared with sham control (Group 2)
during early phase of 2 Gy (Group 3) and 8 Gy (Group 4) post-irradiation. Data were expressed as means values ± SEM.

159 flow cytometry (FACSVerse II, BD Biosciences, San concentration of cytokines within TME were compared 187

160 Jose, CA, USA) at Faculty of Pharmacy, Universiti across multiple groups by Kruskal-Wallis and Dunn’s 188

161 Teknologi MARA. The FACS data were then analysed post-tests with p 6 0.05 is considered as significant. 189

162 using FlowJo v10. Data were expressed as means values ± standard error 190

mean (SEM) of at least three independent experiments. 191

163 2.8. Multiplex assay

164 Protein was extracted and estimated using Radioim- 3. Results 192

165 munoprecipitation Assay (RIPA, Sigma-Aldrich Solu-

166 tion, Darmstadt, Germany) Buffer and PierceTM BCA 3.1. No difference in tumour growth during the early 193

167 Protein Assay Kit (Thermo-Scientific), respectively. A phase post-irradiation in both treatment groups 194

168 70 µL of 5.5 ± 0.5 mg/ml protein from each sample was

169 loaded (in duplicate) into each 96 well plate that con- Gradual increment in tumour volume in 2 Gy and 195

170 tained a mixture of fluorescent-coded magnetic beads 8 Gy treated-groups 3 and 4, respectively were observed 196

171 and pre-coated with 25 analytes i.e., CCL11/eotaxin, on days 5th , 7th and 9th post-inoculations. Sham control 197

172 IFN-γ, IL-1a, IL-1b, IL-3, IL-5, IL-6, IL-7, IL-9, IL- group was sacrificed on day 10th post-inoculation, while 198

173 10, IL-12p40, IL-13, IL-15, IL-17, IP-10, KC, MCP, the other treated groups were continuedly exposed to 199

174 MIP-1a, M-CSF, VEGF, CCL5/RANTES, TNF-α, LIF, irradiation. No significant increment in tumour volume 200

175 LIX, and GM-CSF (25-Plex Assay Kit, Merck Milli- between Groups 3 and 4 at both 2nd and 4th days post- 201

176 pore, Darmstadt, Germany). After overnight incubation irradiation was observed in the early phase, before sacri- 202

177 at 4◦ C, The signal from each cytokines was quantified ficed. Similarly, no significant difference was observed 203

178 using MAGPIX R equipped with Bio-Plex Manager between Groups 3 and 4, despite a lower mean value 204

179 software V.4.0 (Bio-Rad Laboratories, Hercules, CA, of tumour growth in Group 3 (930.88 ± 74.78 mm3 ) 205

180 USA) available at University Malaya (Malaysia). The compared to Group 4 (1083.51 ± 78.17 mm3 ) (Fig. 2). 206

181 concentration of each cytokines was determined based

182 on standard curves prepared earlier. 3.2. No significant difference in the mean life span of 207

post-irrradiation mice in the acute phase in both 208

183 2.9. Statistical analysis treatment groups 209

184 Statistical analysis was performed using GraphPad The mean life span of mice irradiated by 2 Gy 210

185 Prism (GraphPad Software, San Diego, CA, USA). gamma-ray (Group 5) was 20 ± 1.46 days, with re- 211

186 Changes in blood analysis, immune population, and spective minimum and maximum lifespans of 16 and 212
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N. Hasan et al. / Immune cells and cytokines induced by gamma-ray 5

3.4. Gamma-ray irradiation induces changes to 250

lymphocytes and neutrophils within TME in the 251

early phase 252

The immune cells within TME were isolated and 253

measured acccording to flow cytometry gating strat- 254

egy by Ibahim et al. (2022) based on previous stud- 255

ies published by Reichman et al. [28] and Yu et 256

al. [29]. Immune cells within tumour sections in con- 257

trol and radiation-treated groups in the early and acute 258

Fig. 3. The mean life span of mice in respective Groups 5 and 6, phases were isolated using the CD45 marker. The pro- 259
after treatment with a single dose of either 2 Gy or 8 Gy gamma cess continued with the isolation and measurement of 260
irradiations in the acute phase.
different subpopulations of immune cells, including 261

213 24 days. While, the mean lifespan of 8 Gy irradiated myeloid neutrophils (CD45+ Ly6CG+ ), lymphocytes 262

214 mice (Group 6) was 18.67 ± 0.84 days, with respective (Ly6G− CD11b− ), T-cells (CD11b− IA/IE), B-cells 263

215 minimum and maximum lifespans of 16 and 20 days. (CD11b− IA/IE+ ), eosinophils (Ly6G− CD11b+ IA/IE− 264

216 No significant difference was obtained between 2 Gy Siglec F+ ), natural killer cells, monocytes (Ly6G− 265

217 and 8 Gy both groups after 15 to 30 days of post- CD11b+ IA/IE− Siglec F− ), dendritic cells and 266

218 inoculation in the acute phase (Fig. 3). macrophages (Ly6G− CD11b+ IA/IE+ ). The percent- 267

age of these cells infiltrating TME in sham con- 268

219 3.3. Differential responses to irradiation doses on trol (Group 2) and irradiation-treated groups in early 269

220 different types of white blood cells in early and (Groups 3 and 4) and acute (Groups 5 to 6) phases are 270

221 acute phases presented in Table 2. 271

Sham control group has the highest percentage of 272

222 The total white blood cell (TWBC) count, absolute total immune cells (44.38 ± 4.28 percent) compared to 273

223 subsets counts, and percentage counts in negative con- all radiation-treated groups. In comparison to systemic 274

224 trol (Group 1), sham control (Group 2) and mouse- circulation (Table 1), neutrophils dominated within 275

225 bearing tumour models in early (Groups 3 and 4) and TME in sham control (63.05 ± 5.61 percent), followed 276

226 acute (Groups 5 to 6) phases are presented in Table 1. by monocytes and natural killer cells (15.70 ± 4.05 277

227 There was no significant difference in white blood cell percent), dendritic cells and macrophages (10.83 ± 278

228 (WBC) counts and percentages between Groups 1 and 2.55 percent), lymphocytes (7.21 ± 2.79 percent), and 279

229 2. Irradiations of 2 Gy (p < 0.01) and 8 Gy (p < 0.001) eosinophils (1.64 ± 0.75 percent) (Table 2). 280

230 reduced the mean of TWBC in Groups 3 and 4 com- Contrary to increment in lymphocytes especially T- 281

231 pared to Group 2 in the early phase. However, these cells, (p < 0.01), neutrophils’ was decreased (p < 0.01) 282

232 effects were only temporary since no significant differ- in early response to 2 Gy irradiation (Group 3) com- 283

233 ence was obtained between both Groups 5 and 6 with pared to sham control. Similar changes in lymphocytes 284

234 Group 2 in the acute phase. (dominated by T-cells, p < 0.001) and neutrophils’ 285

235 During the early phase, both basophils’ absolute (p < 0.05) to the early response of 8 Gy irradiation 286

236 count (p < 0.05) and percentage count (p < 0.05) were (Group 4) was obtained. Total immune cell population 287

237 decreased in response to both irradiation doses (Groups (p < 0.05), dendritic cells (p < 0.01) and macrophages 288

238 3 and 4) compared to sham control (Group 2). Similar (p < 0.01) was lower in Group 4 compared to sham 289

239 finding was obtained in eosinophils’ percentage count control (Table 2). 290

240 (p < 0.01). The suppression to neutrophils’ (p < 0.01) As expected, higher changes in the differences across 291

241 and lymphocytes’ (p < 0.001) absolute counts were study groups compared to systemic circulation was ob- 292

242 only obtained at 8 Gy (Group 4) compared to sham served in the acute phase. Neutrophils, B-cells, mono- 293

243 control. On the contrary, monocytes’ percentage count cytes and natural killer cells in Group 5 remained higher 294

244 was higher (p < 0.01) compared to sham control. (p < 0.05) than sham control in response to 2 Gy ir- 295

245 As expected, lymphocytes’ absolute count and radiation. Except for increment in eosinophils (p < 296

246 eosinophils’ percentage were decreased (p < 0.05) after 0.05), total immune cell populations, dendritic cells and 297

247 2 Gy and 8 Gy irradiations in the acute phase (Groups 5 macrophages in Group 6 remained lower (p < 0.001) 298

248 and 6) compared to sham control. However, no signifi- than sham control in response to 8 Gy irradiation (Ta- 299

249 cant difference was observed in the other parameters. ble 2). 300
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6 N. Hasan et al. / Immune cells and cytokines induced by gamma-ray

Table 1
The total white blood cell count, absolute subsets counts, and percentage counts in negative control, sham control and mouse-bearing tumour
models obtained from blood circulation
Healthy
(Group 1:
Parameters Negative Mouse-bearing tumour model
control)
(n = 6)
Non-radiate
(Group 2: Early phase Acute phase
Sham control) (96 hours post-irradiation) (15 to 30 days post-irradiation)
(n = 4)
Group 3: Group 4: Group 5: Group 6:
2 Gy (n = 6) 8 Gy (n = 5) 2 Gy (n = 5) 8 Gy (n = 3)
Total white blood cell (103 /µL) 6.96 ± 1.40 5.27 ± 0.51 2.41 ± 0.15∗∗ 0.68 ± 0.11∗∗∗ 3.95 ± 0.86 5.53 ± 3.46#
Neutrophils (103 /µL) 0.31 ± 0.07 0.39 ± 0.04 0.20 ± 0.06 0.09 ± 0.01∗∗ 1.11 ± 0.68# 0.42 ± 0.19#
Neutrophils (%) 4.72 ± 0.55 7.70 ± 1.50 5.32 ± 1.33 14.38 ± 2.69 13.66 ± 1.33∗## 9.23 ± 1.47
Lymphocytes (103 /µL) 5.66 ± 1.06 3.65 ± 0.26 1.90 ± 0.12 0.47 ± 0.10∗∗∗ 1.62 ± 0.20∗ 3.57 ± 2.02#
Lymphocytes (%) 82.18 ± 2.62 69.73 ± 2.28 79.25 ± 4.16 65.12 ± 2.87 45.40 ± 6.43∗### 70.73 ± 6.34
Monocytes (103 /µL) 0.19 ± 0.11 0.08 ± 0.01 0.27 ± 0.03 0.12 ± 0.03 0.24 ± 0.18
Monocytes (%) 2.74 ± 1.09 1.60 ± 0.31 11.12 ± 1.60∗∗ 16.28 ± 2.31∗∗∗ 5.48 ± 1.75 3.42 ± 0.65#
Eosinophils (103 /µL) 0.005 ± 0.002 0.01 ± 0 0 0 0.005 ± 0.003 0
Eosinophils (%) 0.08 ± 0.04 0.58 ± 0.38 0∗∗ 0∗ 0.12 ± 0.10 0∗
Basophils (103 /µL) 0.49 ± 0.18 0.91 ± 0.13 0.15 ± 0.07∗ 0.02 ± 0.01∗∗∗ 0.88 ± 0.15# 1.31 ± 1.07#
Basophils (%) 8.05 ± 2.31 17.20 ± 1.66 3.60 ± 0.95∗ 4.22 ± 1.45∗ 24.69 ± 3.95## 24.69 ± 3.95##
Data were expressed as mean values ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 compared to sham control, # p < 0.05; ## p < 0.01;
### p < 0.001 compared to responses between early and acute phases after the same dose of treatment.

301 3.5. Targeted gamma-ray irradiation potentially On the contrary, IL-6 (both doses p < 0.01), GM-CSF 328

302 suppressed pro-tumorigenic cytokines within TME (both doses p < 0.05), VEGF (2 Gy; p < 0.01 and 329

8 Gy; p < 0.001) and IL-7 (2 Gy; p < 0.001 and 8 Gy; 330

303 A total of 25 cytokines-related immune parameters p < 0.05) were decreased compared to sham control. 331

304 were selected and measured to investigate the effect Interestingly, IL-3 (p < 0.05), IL-15 (p < 0.01), KC 332

305 of single dose irradiation in TME. Except for IL-13, (p < 0.001), and LIX (p < 0.01) were decreased in 333

306 the other 24 cytokines were detected with 15 showing Group 5 compared to sham control (Table 3a). 334

307 significant changes across the groups (Table 3a), and

308 9 without changes (Table 3b). IL-17 (p < 0.05), IL-1b
309 (p < 0.05), and CCL11 (p < 0.05 and p < 0.0001, 4. Discussion 335

310 respectively) were increased to early response of 2 Gy

311 and 8 Gy gamma-ray irradiation in Groups 3 and 4, Based on the Mouse Phenome Database (MPD) re- 336

312 respectively compared to sham control (Group 2). In- ports, the healthy 8-week-old female BALB/c mice used 337

313 crement to RANTES (p < 0.01) and TNF-α (p < 0.01) in this study have similar total white blood cell count 338

314 were also obtained in response to 8 Gy irradiation in and lymphocytes’ percentage. However, basophils have 339

315 Group 4 compared to sham control. In contrary, VEGF a twenty-fold greater percentage compared to the stan- 340

316 (p < 0.05 and p < 0.001, respectively), IL-6 (p < 0.01 dard [30]. Despite identical strain and age, other factors 341

317 and p < 0.05, respectively), and GM-CSF (p < 0.001) such as environmental, dietary, and handling variables 342

318 in Groups 3 and 4 decreased compared to sham con- influenced the physiological characteristics, including 343

319 trol. Decrement in KC (p < 0.0001) in Group 3, and immunological state of the mice [31]. 344

320 increment in IL-7 (p < 0.0001) and IL-15 (p < 0.05) Generally, uncontrolled growth of either cells or tis- 345

321 in Group 4 was obtained in comparison to sham control sues or tumour resulted from failure of innate and adap- 346

322 (Table 3a). tive immune responses leading to various homeosta- 347

323 Similarly, in the acute phase of post-irradiation IL-17 sis abnormalities. On day 5th after inoculation with 348

324 (both doses p < 0.05), IL-1b (2 Gy; p < 0.001, 8 Gy; EMT6 cells, tumour-bearing mouse began to exhibit 349

325 p < 0.01), eotaxin (2 Gy; p < 0.01, 8 Gy; p < 0.05) and signs of tumour progression. The absolute count of sys- 350

326 TNF-α (both doses p < 0.01) continued to elevate in temic eosinophils and basophils increased by day 9th 351

327 Groups 5 and 6, respectively compared to sham control. post-inoculation, although lymphocytes and monocytes 352
353
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Table 2
The percentage of immune cells presence within TME in response to 2 Gy and 8 Gy gamma-ray irradiations duing early and acute phases
Group 2:
Parameter Sham control Radiation treatment groups
(n = 4)
Early phase (96 hours Acute phase (between
14/07/2023; 10:18

post-irradiation) 15 to 30 days post-inoculation)

Group 3: Group 4: Group 5: Group 6:
2 Gy (n = 6) 8 Gy (n = 5) 2 Gy (n = 5) 8 Gy (n = 3)
Mean ± S.E.M Min Max Mean ± S.E.M Min Max Mean ± S.E.M Min Max Mean ± S.E.M Min Max Mean ± S.E.M Min Max
CD45 44.38 ± 4.28 32.20 64.80 35.08 ± 3.23 24.30 46.80 35.24 ± 2.62∗ 24.50 55.90 37.72 ± 1.61 30.40 47.10 30.71 ± 1.72∗∗∗ 22.30 39.40
Neutrophils 63.05 ± 5.61 29.10 71.10 27.20 ± 3.70∗∗ 11.70 37.90 28.93 ± 4.24∗∗ 9.41 70.50 36.68 ± 4.46∗ 27.40 56.90 51.63 ± 3.98### 39.20 68.00
Lymphocytes 7.21 ± 2.79 1.87 36.19 42.33 ± 4.90∗∗ 31.77 65.30 48.22 ± 4.80∗∗∗∗ 12.04 85.17 22.57 ± 1.68 16.38 28.91 13.89 ± 1.96#### 6.09 23.74
B cells 0.24 ± 0.07 0.07 2.53 2.47 ± 1.24∗ 0.48 7.00 0.50 ± 0.12 0.06 1.29 1.78 ± 0.76∗ 0.31 5.24 0.15 ± 0.05 0.04 0.49
T cells 6.97 ± 2.73 1.80 12.39 37.42 ± 2.77∗∗ 30.59 48.18 47.72 ± 4.76∗∗∗∗ 11.98 85.00 20.79 ± 1.15 15.78 23.67 13.73 ± 1.95#### 5.85 23.25
Eosinophils 1.64 ± 0.75 0.26 8.97 1.79 ± 0.28 0.90 2.80 2.31 ± 0.22 0.84 3.35 1.46 ± 0.30 0.71 2.62 3.78 ± 0.46∗,# 2.17 5.34
Monocytes & NK 15.70 ± 4.05 1.20 27.21 18.15 ± 3.40 3.46 26.54 15.75 ± 1.84 3.34 28.45 28.08 ± 3.40* 16.56 41.50 24.30 ± 2.40 16.80 33.48
DC & macrophage 10.83 ± 2.55 5.76 37.05 9.11 ± 1.65 3.94 15.48 3.80 ± 0.79∗∗ 0.54 10.25 7.90 ± 2.17 1.41 16.65 3.92 ± 0.63∗ 1.73 6.87
Data were expressed as means values ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 compared to Sham control, # p < 0.05; ## p < 0.001, ### p < 0.001; #### p < 0.0001 compared to
responses between early and acute phases after the same dose of treatment.
N. Hasan et al. / Immune cells and cytokines induced by gamma-ray
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Table 3a
Targeted gamma-ray irradiation of 2 Gy and 8 Gy induce changes to cytokines within TME in mouse-bearing tumour model
Group 2:
Cytokine Sham control Radiation treatment groups
(n = 4)
Early phase (96 hours Acute phase (between
post-irradiation) 15 to 30 days post-inoculation)
Group 3: Group 4: Group 5: Group 6:
2 Gy (n = 6) 8 Gy (n = 5) 2 Gy (n = 5) 8 Gy (n = 3)
14/07/2023; 10:18

Mean ± S.E.M Min Max Mean ± S.E.M Min Max Mean ± S.E.M Min Max Mean ± S.E.M Min Max Mean ± S.E.M Min Max
Eotaxin 368.1 ± 70.7 221.6 697.2 857.0 ± 162.9∗ 226.8 1631.0 1039.0 ± 61.07∗∗∗∗ 623.4 1296.0 871.1 ± 50.35∗∗ 590.9 1049.0 742.2 ± 95.14∗ 351.5 1061.0
∗∗∗ ∗∗∗ ∗ ∗
GM-CSF 2039 ± 110 1769 2550 1084 ± 184.5 234 1811 1078 ± 191 298 2173 1345 ± 183.2 670 2524 1387 ± 207.4 445 2362
IFN-γ 17.4 ± 2.4 7.8 28.0 19.5 ± 1.5 10.9 25.5 23.2 ± 2.5 10.4 35.1 14.7 ± 2.0 4.0 30.5 15.3 ± 2.458# 6.6 30.2
IL-15 318.7 ± 70.0 92.8 647.1 328.7 ± 86.85 61.7 721.2 103.3 ± 6.5∗ 74.6 133.6 86.8 ± 10.5∗,# 55.0 131.3 181.0 ± 32.1 53.1 399.9
IL-17 0.8 ± 0.1 0.4 1.4 2.0 ± 0.4222∗ 0.7 3.7 2.2 ± 0.4812∗ 0.1 5.2 2.2 ± 0.403∗ 0.3 3.8 2.3 ± 0.4784∗ 0.5 5.9
∗ ∗
IL-1b 5.8 ± 0.9 3.3 11.3 16.1 ± 4.23 3.8 31.7 15.7 ± 2.547 4.0 30.3 32.8 ± 6.389∗∗∗ 6.2 57.0 10.5 ± 2.911∗∗ 3.4 27.9
∗,#
IL-3 21.0 ± 2.4 12.1 31.7 27.9 ± 6.8 13.5 59.4 29.5 ± 4.7 8.6 46.9 10.5 ± 1.2 3.5 15.7 29.4 ± 5.163 7.1 58.8
∗∗ ∗ ∗∗
IL-6 119.2 ± 12.2 72.9 160.3 67.3 ± 13.48 31.3 142.5 83.5 ± 12.98 35.6 153.6 65.5 ± 3.898 44.2 83.6 64.5 ± 9.958∗∗ 15.8 100.3
∗∗∗,# ∗,##
IL-7 95.1 ± 9.4 50.3 126.6 63.6 ± 9.6 12.5 108.6 24.7 ± 3.002∗∗∗∗ 13.0 37.3 36.5 ± 2.75 24.6 52.5 53.0 ± 4.6 33.6 80.4
IL-9 327.4 ± 51.1 183.8 546.6 223.6 ± 21.4 100.8 347.8 592.3 ± 137.1 210.0 1227.0 507.8 ± 56.39### 203.5 759.5 313.2 ± 50.8 194.9 747.0
∗∗∗∗ ∗∗∗
KC 9725 ± 1491 3214 14199 1731 ± 251.5 1046 3104 6403 ± 775 2738 9094 2277 ± 559.8 1043 5692 7364 ± 1546 2908 15909
LIX 687.4 ± 81.0 315.6 908.9 970.2 ± 277.5 130.5 2411.0 934.1 ± 273.5 183.8 2451.0 203.6 ± 29.05∗,# 102.8 291.3 675.6 ± 80.5 333.3 998.0
RANTES 5.4 ± 0.7 2.6 8.4 7.7 ± 1.3 1.4 14.9 12.6 ± 1.34∗∗ 5.4 17.7 6.8 ± 1.2 0.9 13.7 5.8 ± 1.387#### 2.0 16.9
TNF-α 7.6 ± 1.3 0.7 11.1 20.7 ± 7.5 1.8 49.8 26.7 ± 3.553∗∗ 12.9 50.0 45.0 ± 10.87∗∗ 0.6 93.0 45.5 ± 13.36∗∗ 2.1 105.4
∗ ∗∗∗∗ ∗∗∗ ∗∗
VEGF 4151 ± 239 3215 5036 3029 ± 263.6 2077 4481 2087 ± 202.3 1047 2612 2396 ± 264.8 1022 3616 2624 ± 142.3 1963 3658
N. Hasan et al. / Immune cells and cytokines induced by gamma-ray

Data expressed as means values ± SEM. ∗ significant with p < 0.05; ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 compared to sham control group, # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001
compared to responses between early and acute phases after the same dose of treatment.
File: cbm–1-cbm220268.tex; BOKCTP/xjm p. 8
 Galley Proof

Table 3b
Targeted gamma-ray irradiation of 2 Gy and 8 Gy have no effects on following cytokines within TME in mouse-bearing tumour model
Group 2:
Cytokine Sham control Radiation treatment groups
(n = 4)
Early phase (96 hours Acute phase (between
14/07/2023; 10:18

post-irradiation) 15 to 30 days post-inoculation)

Group 3: Group 4: Group 5: Group 6:
2 Gy (n = 6) 8 Gy (n = 5) 2 Gy (n = 5) 8 Gy (n = 3)
Mean ± S.E.M Min Max Mean ± S.E.M Min Max Mean ± S.E.M Min Max Mean ± S.E.M Min Max Mean ± S.E.M Min Max
IL-10 15.3 ± 2.7 7.7 28.1 15.2 ± 1.3 7.2 20.7 18.4 ± 2.3 11.0 28.4 11.5 ± 1.0 5.7 16.0 14.3 ± 1.7 7.3 23.0
IL-12p40 5.8 ± 0.7 2.5 9.1 5.8 ± 1.1 1.3 9.4 5.7 ± 0.9 1.5 9.6 6.1 ± 0.8 2.8 10.8 8.2 ± 1.5 2.0 16.0
IL-1a 99.4 ± 21.2 37.4 191.1 99.9 ± 22.3 37.9 217.4 120.6 ± 17.8 37.5 200.9 157.4 ± 16.4 99.1 288.7 106.4 ± 25.0 11.3 301.5
IL-5 4.6 ± 0.4 3.3 6.0 5.5 ± 0.9 1.5 9.9 6.3 ± 0.7 3.0 10.5 4.9 ± 0.8 2.3 10.7 5.4 ± 0.7 2.2 9.5
IP-10 1565.0 ± 233.7 773.1 2455.0 1752.0 ± 348.6 438.2 4338.0 1577.0 ± 139.7 756.3 2191.0 1109.0 ± 128.2 364.7 1717.0 1208.0 ± 125.4 429.5 1888.0
LIF 1747.0 ± 101.6 1414.0 2175.0 1634.0 ± 129.4 1141.0 2277.0 1585.0 ± 95.0 1226.0 2480.0 1484.0 ± 68.3 1040.0 1776.0 1479.0 ± 98.7 1051.0 2040.0
MCP 2719.0 ± 160.5 2197.0 3541.0 2065.0 ± 262.2 862.2 3270.0 3195.0 ± 298.8 1643.0 5087.0 2111.0 ± 248.8 1157.0 3766.0 2755.0 ± 323.8 1014.0 4655.0
M-CSF 822.9 ± 81.3 598.4 1178.0 629.1 ± 135.5 88.8 1339.0 880.0 ± 75.0 603.1 1477.0 595.9 ± 55.3 289.7 872.6 810.2 ± 113.5 231.5 1235.0
MIP 278.3 ± 42.4 158.7 464.4 219.3 ± 42.7 30.5 422.6 361.9 ± 75.0 77.5 734.6 315.0 ± 66.5 129.8 692.1 269.6 ± 60.0 38.1 537.5
Data expressed as means values ± SEM.
N. Hasan et al. / Immune cells and cytokines induced by gamma-ray
9
File: cbm–1-cbm220268.tex; BOKCTP/xjm p. 9
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10 N. Hasan et al. / Immune cells and cytokines induced by gamma-ray

354 count decreased without significant difference in com- phocytes, neutrophils, and basophils were radiosensi- 405

355 parison to healthy mice. Tumour progression also al- tive [49,50]. Accordingly, an increment in the percent- 406

356 tered immune cell percentage with significant eleva- age of monocytes by 10th folds compared to untreated 407

357 tion in eosinophil’s percentage. Tumour development tumour in early response to irradiation was obtained in 408

358 might be decelerated or accelerated depending on the the present study. A variety of malignancies including 409

359 percentage of eosinophils present [32]. Despite several breast cancer, may be predicted by the total white blood 410

360 investigations, the involvement of eosinophils in cancer cell count and its components, especially by the ratio 411

361 remains unclear due to different contradictory tumori- of neutrophils to lymphocytes (NLR) [51–53]. How- 412

362 genic activities obtained from various cells [11,33,34]. ever, direct interpretation of NLR usage in preclinical 413

363 Alteration within microenvironment’s residence was mice model should be cautious as lymphocytes domi- 414

364 expected as the tumour was directly exposed to gamma- nated their systemic circulation rather than neutrophils. 415

365 ray irradiation. Similarities in irradiation-induced im- Briefly, a transient immune suppression effect from ir- 416

366 munosuppression has been established [35–37]. But radiation justified radiotherapy’s usage during the pan- 417

367 presently, no significant difference was obtained in demic since its advantages and hazards, such as non- 418

368 tumour development between irradiation-treated and invasive and short-term immune suppression, outweigh 419

369 sham control groups. A declined in the number of sys- those of other treatment methods that potentially caused 420

370 temic TWBC compared to untreated group in early re- severe infection [4,54]. 421

371 sponse to both irradiation doses indicated that irradia- The main effect of high-energy gamma-ray irradia- 422

372 tion as the caused for systemic effect. The indication tion on TME was the destruction of tumour endothe- 423

373 is concluded based on our experimental setting where lial cells involved in triggering an immune response. 424

374 single targeted irradiation was exposed specifically to TME consisted of various cells, including blood ves- 425

375 tumour site at hind leg, as well as the presented impact sels, connective tissues, immune cells, and epithelial 426

376 on TWBC in the systemic circulation [38]. cells that emitted a variety of signalling molecules, in- 427

377 Early immunomodulatory effects are dose- cluding cytokines which governed the progression and 428

378 dependent [39,40], since less alteration in the systemic growth of cancer. Certain cytokines, chemokines and 429

379 immune population observed with considerably lower growth factors influenced the immune response within 430

380 dosage of 2 Gy compared to 8 Gy gamma-ray irradia- TME [55–58]. Various factors, including dosages and 431

381 tion. In this study, systemic modulation was presented fractions of radiation treatments, potentially altered 432

382 in certain immune cells in either their absolute num- these mediators. Thus, the impact would affect either 433

383 ber or/and percentages. It can be seen in this study for the activation or suppression of immune cells involved 434

384 both the percentages and absolute numbers of systemic in either pro- or anti-tumorigenic activities [59]. 435

385 eosinophils and basophils irradiated with 8 Gy gamma- Our experimental design enabled the isolation and 436

386 ray were significantly reduced, however, only the ab- measurement of the most common immune cells infil- 437

387 solute numbers were reduced for neutrophils and lym- trating TME. In contrast to eosinophils, the involvement 438

388 phocytes. Since eosinophil changes are low, several hu- of neutrophils, macrophages, myeloid-derived suppres- 439

389 man studies reported the changes in eosinophil and neu- sor cells (MDSCs), natural killer cells, and both B- 440

390 trophil as eosinophil to lymphocyte ratio (ELR) or neu- and T-lymphocytes in cancer progression and suppres- 441

391 trophil to lymphocyte ratio (NLR) and have been used sion have been extensively addressed [14,60,61]. Our 442

392 as a prognosis marker for inflammation event [41–44]. findings were consistent with prior studies, which re- 443

393 However, in this study, the ratio of ELR obtained in nor- ported neutrophils and macrophages as the most preva- 444

394 mal mice is too large (1:1132) while the other radiation- lent immune cells penetrating tumours [62,63]. Tumour- 445

395 treated groups have a 0 value that is unable to get the associated neutrophils (TANs), a term for neutrophils 446

396 ratio. Changes in eosinophils absolute count appeared invading TME, appeared to have both pro- and antitu- 447

397 to vary depending on species and site of irradiation mour effects [62,64]. 448

398 exposure [11,45]. One of neutrophils’ anticancer effects was releasing 449

399 Lymphoid system’s vulnerability to ionising radia- mediators involved in the recruitment and activation of 450

400 tion is well known to cause lymphopenia [46,47]. How- other immune cells under innate or adaptive immune 451

401 ever, our study suggested basophils as more likely to systems [10,65]. In contrast, pro-inflammatory medi- 452

402 be impacted. The effects of irradiation on basophils ators, including IL-6 and IL-1 were also secreted by 453

403 in both human and mouse models were unclear [48]. TANs. In our study, the early response to single tar- 454

404 Monocytes were the most radioresistance, while lym- geted irradiation resulted in a substantial drop in IL-6 455
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N. Hasan et al. / Immune cells and cytokines induced by gamma-ray 11

456 level compared to sham control. Notably, a decreased in ous study by Ibahim et al. [25] that reporting an in- 507

457 TANs was parallel with a reduction in IL-6, but not IL-1 crement of eosinophil-related gene expression, CCL11 508

458 in the irradiation-treated groups, thus suggesting pro- or eotaxin after exposure to 7 Gy gamma-ray irradia- 509

459 tumour stimulators were suppressed in early response tion. Nine of the selected cytokines exhibited substan- 510

460 to irradiation. tial alterations in response to both gamma-ray irradi- 511

461 The released of IL-6 was also contributed by cancer- ation dosages of 2 Gy and 8 Gy was presented Ta- 512

462 associated fibroblasts (CAFs) which was crucial for ble 3a. Three cytokines were associated with response 513

463 cancer progression [66–68]. An increased in CAFs ac- to 2 Gy irradiation, but only RANTES was associated 514

464 tivity accelerated tumour proliferation, angiogenesis, with response to 8 Gy irradiation. RANTES/CCL5, 515

465 metastasis, and survival, as well as poor chemother- CCL11/eotaxin, VEGF, GM-CSF, IL-1, IL-6, IL-7, 516

466 apy response [67]. On the other hand, suppressing IL- IL-15, IL-17, and TNF were considerably altered in 517

467 6 expression was associated with CAFs inhibiting the response to 8 Gy irradiation and corresponded with 518

468 spreading of certain cancers [68]. Although the exis- TATE. These chemokines were also responsible for 519

469 tence of CAFs was not determined in this study, the anti-angiogenesis, thus collectively serving as an anti- 520

470 lower level of IL-6 in irradiation groups denoted the tumorigenic factor because increment in CCL11 ex- 521

471 destruction of CAFs and consequently suggesting an pression favourable to the survival of cancer patients by 522

472 anti-tumour impact. The released of IL-6 from CAFs metastasis inhibition [80,81]. 523

473 interfered with the action of immune surveillance, such Damage to tumour vascularization was another well- 524

474 as hindering tumour infiltrating lymphocytes (TILs) and documented consequence of irradiation on TME and 525

475 increasing tumour survival [66]. has been extensively [82]. Deficiencies in blood flow 526

476 Interestingly, decreased in TANs and IL-6 occurred and oxygen supply led to a hypoxic microenvironment 527

477 concurrently with elevation of TILs population within within the tumour. Thus, hypoxic situation stimulated a 528

478 TME. Growing evidence suggested certain TILs sub- rise in the expression of VEGF by tumour tissue. As an 529

479 types reflected different prognostic significance in vari- important player in tumour angiogenesis, VEGF helps 530

480 ous cancers [69–71]. TILs mainly composed of T-cells delivered nutrients and oxygen necessary for tumour 531

481 consisting of different subsets, including regulatory T growth and development [83,84]. Astonishingly, we 532

482 cells (Tregs) or CD4+ and cytotoxic T lymphocytes found decrement in VEGF in radiation-treated groups 533

483 (CTLs) or CD8+ were important in host’s immune slowing tumour angiogenesis. A decreased in VEGF 534

484 response to tumours [72–74]. On the other hand, an- expression resulted from irradiation exposure suggested 535

485 other subtype of Tregs that expressed Foxp3 recep- an anti-tumorigenic activity. This was confirmed by 536

486 tors were thought to dampen anti-tumour immune re- metanalysis study demonstrating a connection between 537

487 sponses [66,75]. poor prognosis with high expression of VEGF among 538

488 Another result from single targeted irradiation of ovarian cancer patients [85]. Apart from that, VEGF can 539

489 2 Gy in parallel with reduction in TANs was a decreased dampen an immune response by decreasing the matura- 540

490 in macrophages and dendritic cells’ populations. As tion of anti-tumorigenic immune cells such as dendritic 541

491 with TANs, there are two subtypes of tumour-associated cells and increasing pro-tumorigenic cell types, such as 542

492 macrophages (TAM) responsible for antitumour and T-regulatory cells, MDSC, and TAMs [86]. 543

493 pro-tumour effects: type 1 macrophage (M1) and type-2 The role of different subtypes of IL-17 in both pre- 544

494 macrophage (M2) [76,77]. M2 was associated with the clinical and clinical breast cancer experiments was 545

495 expression of numerous pro-tumour mediators, includ- discussed Fabre et al. [87]. In summary, IL-17 was 546

496 ing VEGF and IL-6 [78]. The inhibition of both cy- suggested as the new target in cancer therapy as 547

497 tokines suggested the treatment given induced suppres- it promoted tumour progression [85,88], angiogene- 548

498 sion of M2 rather than M1. In addition, the rise of TNF sis, and resistance toward anti-tumour immunity, de- 549

499 i.e., a cytokine released by M1 [79], provided addi- spite demonstrating anti-tumourigenic effect [87,89]. 550

500 tional evidence for the notion that targeting gamma-ray The correlation between IL-17 with TNF-a in tu- 551

501 irradiation at 2 Gy induced repression of pro-tumour mour progression and systemic inflammation were dis- 552

502 activity. cussed. Depending on the IL-17 subtypes, they either 553

503 An interesting finding in the acute response to single served as pro-tumour cytokines leading to the pro- 554

504 targeted 8 Gy was an increment of eosinophils within duction of other pro-tumorigenic cytokines [55,88,90] 555

505 TME, known as tumour-associated tissue eosinophilia and/or anti-tumour cytokines inducing immune recruit- 556

506 (TATE). This discovery was consistent with a previ- ment [88,91,92]. However, the suppressive effects on 557
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12 N. Hasan et al. / Immune cells and cytokines induced by gamma-ray

558 other pro-tumorigenic cytokines i.e., IL-6, GM-CSF, for 36 cancers in 185 countries, CA: A Cancer Journal for 596

559 and VEGF obtained in our initial study suggested IL-17 Clinicians 71 (2021), 209–249. 597
[3] Z. Momenimovahed and H. Salehiniya, Delay in the diagnosis 598
560 acted synergistic with TNF-a cytokine post-radiation of breast cancer during coronavirus pandemic, EXCLI Journal 599
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563 Different doses of single targeted gamma-ray irradi- treated with hypofractionated radiotherapy, Indian Journal of 606

564 ation were proposed to cause temporary changes in the Cancer 57 (2020), 423–427. 607
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565 immune population within TME, which differ in im- A. Di Pilla, L.A. Ursini, M. Taraborrelli, A. Vinciguerra, A. 609
566 mune population in the systemic blood circulation and Augurio, M. Di Tommaso, M. Trignani, M. Nuzzo, M.D. Falco, 610
567 mainly correlated with suppression of pro-tumorigenic A. De Nicola, N. Adorante, F. Patani, G. Centofanti, L. Gas- 611

568 cytokines. Further investigation and a more extended parini, D. Fasciolo, F.C. Di Guglielmo, C. Bonfiglio, M. Bor- 612
gia, G. Caravaggio, S. Marcucci, C. Turchi, D. Mancinelli, S. 613
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571 Conflict of interest and G. Ingrosso, Radiotherapy in the era of COVID-19, Expert 619
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572 The authors have no conflicts of interest to disclose. [8] A. Ejaz, J.S. Greenberger and P.J. Rubin, Understanding the 621
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573 Acknowledgments A.L. Pourzia, S. Schad, S.F. Johnson, R.D. Carrasco, S. Lazo, 625
R.T. Bronson, S.P. Davis, M. Lobera, M.A. Nolan and A. Letai, 626

574 The study was funded by a Fundamental Re- Class IIa HDAC inhibition reduces breast tumours and metas- 627
tases through anti-tumour macrophages, Nature 543 (2017), 628
575 search Grant Scheme, Ministry of Education Malaysia 428–432. 629
576 (FRGS/1/2016/SKK08/UITM/03/2). [10] V. Governa, E. Trella, V. Mele, L. Tornillo, F. Amicarella, E. 630
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577 Author contributions Neutrophils and CD8(+) T Cells Improves Survival in Human 634
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578 Funding acquisition: MJI. [11] X. Yang, L. Wang, H. Du, B. Lin, J. Yi, X. Wen, L. Geng 636

579 Conception: NH, MJI. and X. Du, Prognostic impact of eosinophils in peripheral 637
blood and tumor site in patients with esophageal squamous 638
580 Methodology: NH, NFRS, MJI. cell carcinoma treated with concurrent chemoradiotherapy, 639
581 Interpretation or analysis of data: NH, NAHH, MKAK, Medicine 100 (2021), e24328. 640

582 MJI. [12] N. Sharma, N. Salaria, S. Kumar, N. Thomas, N. Beniwal and 641

583 Preparation of the manuscript: NH, NAHH, MKAK, R. Singh, The role of tumour-associated tissue eosinophilia 642
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584 SBSAF, EO, MJI. cinoma, The Egyptian Journal of Otolaryngology 37 (2021), 644
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586 MKAK, SBSAF, EO, MJI. [13] Z. Wang, B. Chen, Y. Fu, C. Ou, Q. Rong, X. Kong, W. Xu, 646

587 Supervision: NAHH, EO, MJI. Y. Deng, M. Jiang and J. Xie, Eosinophilia and lung cancer: 647
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