Research ArticleExperimental Studies

Carcinogenesis Observed in the Spleens of Balb/c Mice After Head-neutron Irradiation

YUKO KINASHI, TAKUSHI TAKATA, YOSHINORI SAKURAI and HIROKI TANAKA
Anticancer Research April 2023, 43 (4) 1455-1461; DOI: https://doi.org/10.21873/anticanres.16294

Abstract

Background/Aim: To investigate the long-term influence of head-neutron irradiation on mice spleens, post-radiation late effects were examined in three types of mice: Balb/c and severe combined immunodeficiency (SCID) mice, which have high radio-sensitivities, and C3H mice. Materials and Methods: Neutron irradiation was performed with the neutron beam of the Kyoto University Research Reactor. Survival fractions and the change in spleen size after head-neutron irradiation were investigated in three different types of mice. Physical condition after neutron irradiation was observed for eighteen months. Results: The onset of primary splenic malignant lymphoma was recognized in many of the Balb/c mice 18 months after head-neutron irradiation. Eight months after head-neutron irradiation, many SCID mice developed an abscess in the part exposed to radiation and spleen swelling. The swollen spleen of SCID mice had hematopoiesis from the marrow. Conclusion: Low energy head-neutron irradiation damages immune organs in radiosensitive SCID and Balb/c mice. A combination of boron neutron capture therapy and immunotherapy may be less toxic than low-energy neutron-irradiation alone.

Key Words:

Neutrons, which have a higher cell-killing effect than X-rays, are used for cancer treatment in boron neutron capture therapy (BNCT). BNCT is based on the following mechanism. Irradiation causes boron-10 to absorb low-energy (below 0.5 eV) neutrons. Subsequent nuclear capture and fission reactions produce alpha (helium-4) particles, and a recoiling lithium nucleus (lithium-7) emits a large energy along a very short path (less than 10 mm). These large energy particles can selectively destroy tumor cells due to the short ranges of the particles. Neutrons have a high relative biological effectiveness (RBE) and a high irradiated cell killing effect on cancer cells. In radiation therapy, many researchers have reported that high-energy neutrons such as fast neutrons cause higher secondary cancer-risk than low LET radiation (1, 2). The late effects of thermal neutrons with low-energy neutrons for patients undergoing BNCT have yet to be fully understood. Clinically, the primary indications for BNCT are high grade gliomas and recurrent tumors of the head and neck region (3-6).

Herein, the late effects for three types of mice are investigated after head-neutron irradiation. The consequences such as survival rate, spleen size, and carcinogenesis depend on the mouse type.

Materials and Methods

Three types of mice. CB17/Icr-Prkdcscid/CrICrIj (severe combined immunodeficiency; SCID), BALB/cAnCrlCrlj (Balb/c), and C3H/HeNCrl (C3H) mice were obtained from Charles River Laboratories (Yokohama, Japan) (Charles River Laboratory Japan was renamed the Jackson Laboratory Japan). Table I lists the characteristics of each type of mouse. Seven-week-old female mice were purchased and bred in mice breeding facilities for five to six weeks.

View this table:
Table I.

Characteristics of the three types of mice*.

Irradiation with Kyoto University Research Reactor (KUR) and thermal neutron fluences. The head was irradiated with KUR operated at a 1 MW output for 60 min. Figure 1 shows a photograph of C3H mice before irradiation at the Heavy Water Neutron Irradiation Facility of KUR. The thermal neutron fluences were measured by gold-foil activation analysis. A thermoluminescence dosimeter measured the gamma-ray dose. The mean±SD of the thermal neutron fluences was 2.3±0.2 (E+12) cm−2. The physical dose for the head was 1.0±0.1 Gy. After neutron irradiation by KUR, the mice were kept in animal breeding facilities, and their physical conditions were observed visually.

Figure 1.
Figure 1.

Photograph of a mouse before irradiation at the Heavy Water Neutron Irradiation Facility of Kyoto University Research Reactor viewed from the irradiated side.

Survival rate and spleen weight. The survival fraction was determined by counting the number of surviving mice irradiated using the KUR neutron beam. As the reduction of the spleen appeared when the body weight began to decrease, spleen weights were measured five months after KUR neutron irradiation using an electric analysis scale (model number HR-251AZ, A&D Japan).

Inspection of the spleen and other tumors. Eight months after head-neutron irradiation, snout subcutaneous tuberosity masses appeared in the part exposed to radiation for SCID mice. Furthermore, swollen spleens were detected during dissection of SCID mice. In Balb/c mice, the onset of abdominal bulges and spleen swelling occurred 18 months after head-neutron irradiation. The abscesses and spleens of SCID mice, and the abdominal tumors and spleens of Balb/c mice were removed. The samples were fixed with a 10% formalin neutral buffered solution (WAKO Fujifilm, Osaka, Japan). The histological section was prepared and dyed. The samples were diagnosed pathologically by the Pathology Center Central Institute for Experimental Animals (Kanagawa, Japan).

Results

Survival fraction after head irradiation with a 1-MW neutron beam. Figure 2 depicts the survival fraction of inbred mice by type after head-neutron irradiation. The survival rate of C3H mice began to decline 12 months after head-neutron irradiation and then stabilized. The survival rate of Balb/c mice began to decline eight months after head-neutron irradiation, and half of the irradiated Balb/c mice died within 15 months of head-neutron irradiation. Previous investigators have reported that the mean of the average lifespan (50% survival rate in days) of radiation-free Balb/c mice was between 720-800 days (~25 months) (7). Hence, head-neutron irradiation shortened the lifespan of Balb/c mice. The survival rate of SCID mice began to decline after five months, and all head-irradiated SCID mice died within 14 months. Other researchers have reported that the average lifespan of radiation-free SCID mice was around 60-70 months (8). Similarly, head-neutron irradiation shortened the lifespan of SCID mice.

Figure 2.
Figure 2.

Survival fraction (SF) after head-irradiation by a 1 MW KUR neutron beam. ● Mean SF of C3H/He mice (n=21). ▲ Mean SF of Balb/c mice (n=20). ■ Mean SF of SCID mice (n=21).

Change in spleen size after head-neutron irradiation. Monthly follow-ups showed that irradiation decreased the splenic weight in SCID and Balb/c mice. Figure 3 compares the splenic weight per body weight four months after head-neutron irradiation with 1 MW KUR to that of the control mice without neutron-radiation exposure. Balb/c mice had the largest splenic weight to body ratio (weight ratio) of the three different types of mice before head-neutron irradiation. Balb/c mice showed the largest reduction in weight ratio after head irradiation. The weight ratio of SCID mice was the smallest before irradiation, and head-neutron irradiation further reduced the weight ratio. Head-neutron irradiation had a negligible impact on the weight ratio in C3H mice.

Figure 3.
Figure 3.

Splenic weight per body weight (g) of the three types of mice before irradiation and three months after head-neutron irradiation. Right stripe of the bar graph is the splenic weight per body weight (g) after head-neutron irradiation, whereas the left stripe is that of the control group without irradiation. Histogram bars show the means±SE for eight animals. (*) Significant decrease in splenic weight per body weight (g) compared to no irradiation group. Significant values of p<0.05 were calculated using Student’s tests.

SCID mice showed an abscess around the snout after head-neutron irradiation. Eight months after head-neutron irradiation, 75% of SCID mice (n-8) developed a subcutaneous tuberosity mass around the snout, which was diagnosed as an abscess at the site of irradiation (Figure 4A). All SCID mice with snout abscesses had a swollen spleen due to inflammatory-responsive hematopoiesis from the marrow (Figure 4B).

Figure 4.
Figure 4.

Subcutaneous nodose and spleen of SCID mice. (A) Subcutaneous nodose organization photograph, which was used for a pathological diagnosis of an abscess. HE stain (left) and Gram stain (right). Left photo shows the granulomatous flame, which has Spindle-Hoeppli material. Right photo shows a Gram-positive bacterial lump. (B) Swollen spleen in a SCID mouse. Polymorph-related hematopoiesis from the marrow in the red palp of the spleen is shown.

Carcinogenesis of the spleen after head-neutron irradiation in Balb/c mice. Some of the surviving Balb/c mice showed an abdominal bulge and became inactive 18-months after head-neutron irradiation. Thus, an autopsy was performed for Balb/c mice 450 days after neutron irradiation. The spleens of five of eight Balb/c mice swelled 18-months after head-neutron irradiation. Table II shows the weight and pathological diagnosis of the five Balb/c mice.

View this table:
Table II.

Weight of enlarged spleen and pathological diagnosis of the Balb/c mouse after head neutron irradiation.

The swollen spleens were diagnosed as malignant lymphoma of a splenic origin. Figure 5 shows the preparation of the splenic malignant lymphoma. A lymph node metastatic focus was detected in two of them, and one of the five Balb/c mice with malignant lymphoma of the spleen also had a benign ovarian tumor.

Figure 5.
Figure 5.

Swollen spleen in a Balb/c mouse. Spleen was diagnosed as malignant lymphoma. Left photo shows splenic swelling with the expansion of the white palp. Right photo shows a follicular hyperplastic focus of the large–small non-inequality consisting of lymphoid cells.

Discussion

Post-radiation late effects were examined as an abscopal effect in the spleen of three different types of mice after head-neutron irradiation. The results depended on the type of mice and can be summarized into four points. First, head-neutron irradiation shortened the lifespan of Balb/c and SCID mice. Second, five months after head-neutron irradiation the spleen size of Balb/c and SCID mice regressed, but that of C3H mice did not change. Third, an abscess formed on the snout of SCID mice, and the spleen swelled due to hematopoiesis of the marrow eight months after head-neutron irradiation. Finally, Balb/c mice showed carcinogenesis of the spleen after head-neutron irradiation.

Other researchers have already examined the survival data of different types of mice after whole-body g-ray or X-ray irradiation (7-13). Thus, the influence of head-neutron irradiation on the survival of the three types of mice can be compared to that of whole-body g-ray or X-ray irradiation. In SCID mice, the average lifespan, which is defined as the 50% survival rate after 1 Gy head-neutron irradiation, was 230 days (33 weeks). Other researchers have reported that the average lifespan of SCID mice was 37.8 and 30 weeks after 1 Gy and 2 Gy whole body g-ray irradiation, respectively (8). Thus, the influence of 1 Gy head-neutron irradiation on survival is similar to 2 Gy whole-body g-ray irradiation of SCID mice.

In Balb/c mice, the average lifespan after 1 Gy head-neutron irradiation was 450 days, with a mean age at death of 534-541 days because Balb/c mice were 1-13 weeks of age at the time of head-neutron irradiation. In a large-scale experiment using Balb/c mice, Storer and coworkers reported that the mean age at death was 521-594 days, depending on the dose rate after 2 Gy whole-body g-ray irradiation (13). The influence of 1 Gy head-neutron irradiation on survival is almost equal to that for 2 Gy whole-body g-ray irradiation of Balb/c mice.

As for C3H mice, a previous study reported that the percent survival after 2 Gy whole-body X-ray irradiation was 77% at 70 weeks of age (12). Our result showed that the percent survival was 85% at 74-75 weeks of age for 1 Gy of head-neutron irradiation. Thus, 1 Gy head-neutron irradiation had a smaller influence on the survival rate compared to that of 2 Gy whole-body g-ray irradiation of C3H mice.

SCID mice have few T cells and defective DNA double strand repair (9, 14). Furthermore, SCID mice are severely deficient in immunoglobulin stem cells, and are susceptible to viral, fungal, and bacterial infections (15). Therefore, an abscess formed at the site of neutron irradiation indicates the onset of an infection. A previous study reported that thymic lymphoma was induced at a high frequency after single whole body g-ray irradiation in SCID mice, but the latency of thymic lymphoma was long (8). In contrast, carcinogenicity of SCID mice was not observed in our study. It is thought that death by infectious disease occurred before carcinogenesis was induced in this study.

Previously, we reported that Balb/c mice have a higher radiosensitivity to whole-body g-ray irradiation than C3H mice (16). This experiment showed that the change in spleen size depends on the type of mouse. Although irradiation caused the involution of the spleen of SCID and Balb/c mice, the spleen size of C3H mice remained unchanged four months after head-neutron irradiation.

The spleen is the largest peripheral lymphoid in mice, and it is thought that lymphocytes are mobilized during infection. According to the hematological changes from radiation therapies in patients with cancer, ionizing radiation suppresses the bone marrow and depletes peripheral lymphocytes (17, 18). A previous study reported that the depression of a nonspecific immune response reflects the cytotoxicity of splenocytes in mice after localized g-ray irradiation to the tumor lesion (19). Therefore, the reduction in the spleen size of SCID and Balb/c mice suggests head-neutron irradiation decreased their immunoreactivity.

The higher ratio of malignant lymphoma in the spleen of Balb/c mice 18-months after head-neutron irradiation was not expected. Storer et al. investigated carcinogenesis in nine types of mice (20). Female Balb/c mice had a higher incidence risk of fatal neoplasms in lung cancer, thymic lymphoma, myeloid leukemia, adrenal cancer, and reticulum cell sarcoma than C3H mice after a single whole body g-ray irradiation up to 2 Gy. Ullrich et al. investigated the carcinogenesis induced in Balb/c mice irradiated with high-LET neutrons using Cf-252 neutron sources (21). Low doses of neutrons induced lung and mammary adenocarcinoma and ovarian tumors. None of the above studies reported splenic malignant lymphoma. Thus, malignant lymphoma appearing at a high ratio in the spleen of Balb/c mice 18-months after head-neutron irradiation is a new discovery.

Many researchers have investigated the abscopal effect of combining radiotherapy and immunotherapy (22). This combination therapy aims to reinforce the curative effect to metastatic foci, which are not the irradiation target. Clinical trials testing the combination of immunotherapy with radiotherapy have indicated that the abscopal effect does not decrease the immune activity in patients with cancer treated with local radiotherapy and immunotherapy (23, 24).

This study demonstrates that local low-energy head-neutron irradiation damages immune organs in radiosensitive SCID and Balb/c mice. A combination of BNCT and immunotherapy may be less toxic than low-energy neutron-irradiation alone. However, we previously reported that the neutron beam irradiation for BNCT induced unrepaired cluster DNA damage more prevalent than gamma irradiation (25). Genetic information about radiosensitivity may be predictive of the effect of combining immunotherapy and BNCT for patients with cancer.

Acknowledgements

This study was supported by JSPS KAKENHI and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant number JP 18K07750).

Footnotes

  • Authors’ Contributions

    YK conceptualized the study and developed the method. TT, YS, HT planned the preparations for the experiment. All Authors have approved the submitted version of the manuscript and agreed to be accountable for the work.

  • Conflicts of Interest

    The Authors declare that there are no conflicts of interest in relation to this study.

  • Received January 5, 2023.
  • Revision received January 19, 2023.
  • Accepted January 20, 2023.

References

  1. ↵
    1. Stricklin DL,
    2. VanHorne-Sealy J,
    3. Rios CI,
    4. Scott Carnell LA and
    5. Taliaferro LP
    : Neutron radiobiology and dosimetry. Radiat Res 195(5): 480-496, 2021. PMID: 33587743. DOI: 10.1667/RADE-20-00213.1
    OpenUrlCrossRefPubMed
  2. ↵
    1. Engels H,
    2. Menzel HG,
    3. Pihet P and
    4. Wambersie A
    : Risk assessment for cancer induction after low- and high-LET therapeutic irradiation. Strahlenther Onkol 175(Suppl 2): 47-51, 1999. PMID: 10394397. DOI: 10.1007/BF03038888
    OpenUrlCrossRefPubMed
  3. ↵
    1. Miyatake SI,
    2. Kawabata S,
    3. Hiramatsu R,
    4. Kuroiwa T,
    5. Suzuki M and
    6. Ono K
    : Boron neutron capture therapy of malignant gliomas. Prog Neurol Surg 32: 48-56, 2018. PMID: 29990973. DOI: 10.1159/000469679
    OpenUrlCrossRefPubMed
    1. Kato I,
    2. Fujita Y,
    3. Maruhashi A,
    4. Kumada H,
    5. Ohmae M,
    6. Kirihata M,
    7. Imahori Y,
    8. Suzuki M,
    9. Sakrai Y,
    10. Sumi T,
    11. Iwai S,
    12. Nakazawa M,
    13. Murata I,
    14. Miyamaru H and
    15. Ono K
    : Effectiveness of boron neutron capture therapy for recurrent head and neck malignancies. Appl Radiat Isot 67(7-8 Suppl): S37-S42, 2009. PMID: 19409799. DOI: 10.1016/j.apradiso.2009.03.103
    OpenUrlCrossRefPubMed
    1. Suzuki M,
    2. Kato I,
    3. Aihara T,
    4. Hiratsuka J,
    5. Yoshimura K,
    6. Niimi M,
    7. Kimura Y,
    8. Ariyoshi Y,
    9. Haginomori S,
    10. Sakurai Y,
    11. Kinashi Y,
    12. Masunaga S,
    13. Fukushima M,
    14. Ono K and
    15. Maruhashi A
    : Boron neutron capture therapy outcomes for advanced or recurrent head and neck cancer. J Radiat Res 55(1): 146-153, 2014. PMID: 23955053. DOI: 10.1093/jrr/rrt098
    OpenUrlCrossRefPubMed
  6. ↵
    1. Dymova MA,
    2. Taskaev SY,
    3. Richter VA and
    4. Kuligina EV
    : Boron neutron capture therapy: Current status and future perspectives. Cancer Commun (Lond) 40(9): 406-421, 2020. PMID: 32805063. DOI: 10.1002/cac2.12089
    OpenUrlCrossRefPubMed
  7. ↵
    1. Storer JB and
    2. Ullrich RL
    : Life shortening in BALB/c mice following brief, protracted, or fractionated exposures to neutrons. Radiat Res 96(2): 335-347, 1983. PMID: 6647762.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Ishii-Ohba H,
    2. Kobayashi S,
    3. Nishimura M,
    4. Shimada Y,
    5. Tsuji H,
    6. Sado T and
    7. Ogiu T
    : Existence of a threshold-like dose for gamma-ray induction of thymic lymphomas and no susceptibility to radiation-induced solid tumors in SCID mice. Mutat Res 619(1-2): 124-133, 2007. PMID: 17397880. DOI: 10.1016/j.mrfmmm.2007.02.028
    OpenUrlCrossRefPubMed
  9. ↵
    1. Custer RP,
    2. Bosma GC and
    3. Bosma MJ
    : Severe combined immunodeficiency (SCID) in the mouse. Pathology, reconstitution, neoplasms. Am J Pathol 120(3): 464-477, 1985. PMID: 2412448.
    OpenUrlPubMed
    1. Lindop PJ and
    2. Rotblat J
    : Life-shortening in mice exposed to radiation: effects of age and of hypoxia. Nature 208(5015): 1070-1072, 1965. PMID: 5870558. DOI: 10.1038/2081070a0
    OpenUrlCrossRefPubMed
    1. Storer JB
    : Radiation resistance with age in normal and irradiated populations of mice. Radiat Res 25: 435-459, 1965. PMID: 14303734.
    OpenUrlPubMed
  12. ↵
    1. Norimura T and
    2. Tsuchiya T
    : Long-term effect of whole-body X-irradiation on cell-mediated immune reaction in mice. J Radiat Res 30(3): 226-237, 1989. PMID: 2681723. DOI: 10.1269/jrr.30.226
    OpenUrlCrossRefPubMed
  13. ↵
    1. Storer JB,
    2. Serrano LJ,
    3. Darden EB Jr.,
    4. Jernigan MC and
    5. Ullrich RL
    : Life shortening in RFM and BALB/c mice as a function of radiation quality, dose, and dose rate. Radiat Res 78(1): 122-161, 1979. PMID: 451140.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Blunt T,
    2. Gell D,
    3. Fox M,
    4. Taccioli GE,
    5. Lehmann AR,
    6. Jackson SP and
    7. Jeggo PA
    : Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse. Proc Natl Acad Sci USA 93(19): 10285-10290, 1996. PMID: 8816792. DOI: 10.1073/pnas.93.19.10285
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Bosma GC,
    2. Custer RP and
    3. Bosma MJ
    : A severe combined immunodeficiency mutation in the mouse. Nature 301(5900): 527-530, 1983. PMID: 6823332. DOI: 10.1038/301527a0
    OpenUrlCrossRefPubMed
  16. ↵
    1. Kinashi Y,
    2. Ono K and
    3. Abe M
    : The micronucleus assay of lymphocytes is a useful predictive assay of the radiosensitivity of normal tissue: a study of three inbred strains of mice. Radiat Res 148(4): 341-347, 1997. PMID: 9339950.
    OpenUrlPubMed
  17. ↵
    1. Jameus A,
    2. Kennedy AE and
    3. Thome C
    : Hematological changes following low dose radiation therapy and comparison to current standard of care cancer treatments. Dose Response 19(4): 15593258211056196, 2021. PMID: 34803549. DOI: 10.1177/15593258211056196
    OpenUrlCrossRefPubMed
  18. ↵
    1. Green DE and
    2. Rubin CT
    : Consequences of irradiation on bone and marrow phenotypes, and its relation to disruption of hematopoietic precursors. Bone 63: 87-94, 2014. PMID: 24607941. DOI: 10.1016/j.bone.2014.02.018
    OpenUrlCrossRefPubMed
  19. ↵
    1. Gerber M,
    2. Dubois JB,
    3. Pioch Y and
    4. Serrou B
    : Effects of localized radiotherapy upon the cellular immune response. Radiat Res 85(2): 390-398, 1981. PMID: 7465789.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Storer JB,
    2. Mitchell TJ and
    3. Fry RJ
    : Extrapolation of the relative risk of radiogenic neoplasms across mouse strains and to man. Radiat Res 114(2): 331-353, 1988. PMID: 3375429.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Ullrich RL,
    2. Jernigan MC and
    3. Storer JB
    : Neutron carcinogenesis. Dose and dose-rate effects in BALB/c mice. Radiat Res 72(3): 487-498, 1977. PMID: 339261.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Ngwa W,
    2. Irabor OC,
    3. Schoenfeld JD,
    4. Hesser J,
    5. Demaria S and
    6. Formenti SC
    : Using immunotherapy to boost the abscopal effect. Nat Rev Cancer 18(5): 313-322, 2018. PMID: 29449659. DOI: 10.1038/nrc.2018.6
    OpenUrlCrossRefPubMed
  23. ↵
    1. Kang J,
    2. Demaria S and
    3. Formenti S
    : Current clinical trials testing the combination of immunotherapy with radiotherapy. J Immunother Cancer 4: 51, 2016. PMID: 27660705. DOI: 10.1186/s40425-016-0156-7
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Golden EB,
    2. Chhabra A,
    3. Chachoua A,
    4. Adams S,
    5. Donach M,
    6. Fenton-Kerimian M,
    7. Friedman K,
    8. Ponzo F,
    9. Babb JS,
    10. Goldberg J,
    11. Demaria S and
    12. Formenti SC
    : Local radiotherapy and granulocyte-macrophage colony-stimulating factor to generate abscopal responses in patients with metastatic solid tumours: a proof-of-principle trial. Lancet Oncol 16(7): 795-803, 2015. PMID: 26095785. DOI: 10.1016/S1470-2045(15)00054-6
    OpenUrlCrossRefPubMed
  25. ↵
    1. Kinashi Y,
    2. Yokomizo N and
    3. Takahashi S
    : DNA double-strand breaks induced by fractionated neutron beam irradiation for boron neutron capture therapy. Anticancer Res 37(4): 1681-1685, 2017. PMID: 28373429. DOI: 10.21873/anticanres.11499
    OpenUrlAbstract/FREE Full Text
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Carcinogenesis Observed in the Spleens of Balb/c Mice After Head-neutron Irradiation
YUKO KINASHI, TAKUSHI TAKATA, YOSHINORI SAKURAI, HIROKI TANAKA
Anticancer Research Apr 2023, 43 (4) 1455-1461; DOI: 10.21873/anticanres.16294
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