Low doses of gamma irradiation potentially modifies immunosuppressive tumor microenvironment by retuning tumor-associated macrophages: lesson from insulinoma
Hridayesh Prakash1,4,*, Felix Klug1, Vinod Nadella3, Varadendra Mazumdar3, Hubertus Schmitz-Winnenthal 2,5 and Liudmila Umansky1
1. Translational Immunology Division, German Cancer Research Centre (DKFZ) and National Centre of Tumor Diseases (NCT), Im-Neuenheimer Feld 460, 69120 Heidelberg, Germany,
2. Department of Visceral Surgery, University Medical Centre, D-69120 Heidelberg, Germany and
3. Laboratory of Translational Medicine, School of life Sciences, University of Hyderabad, India, 500046, India
4. Current affiliation: Laboratory of Translational Medicine, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India
5. Current Affiliation: Klinikum Aschaffenburg-Alzenau, Standort Aschaffenburg, Am Hasenkopf 63739 Aschaffenburg, Germany
*To whom correspondence should be addressed.
Email: email@example.com; Tel: +91-40-66794704; Fax: 0091-40-23011089
Macrophages, by their virtue of functional plasticity, can both control or promote tumor growth. Under the influence of immunosuppressive tumor microenvironment, tumor infiltrating M1 effector macrophages, which drive Th1 responses, support vascular normalization, and improve T-cell recruitment in the process of tumor rejection gets polarized to tumor-promoting and immunosuppressive tumor-associated M2 macrophages (TAM). Their increased density is correlated with poor prognosis in cancer patients. Therefore functional retuning of TAM to an M1 phenotype is paramount for effective immunotherapy against established tumors which could be afforded by systemic application of low dose gamma irradiation in neo adjuvant setting of late-stage human insulinoma (Pan-NET) bearing Rip1-Tag5 mice. Low dose radiation resulted in profound induction of M1-associated effecter cytokines, enhanced tumor infiltration of M1 and concomitant reduction of M2 macrophages in irradiated insulinoma tumors. We also observed significant reduction in the intratumoral as well global reduction of protumorigenic and M2-associated effecter cytokines by gamma irradiation. Most interestingly, presence of peritumoral macrophages was found indispensible for gamma irradiation triggered M1 retuning of insulinoma demonstrating the critical role of M1 effector macrophages for retuning of insulinoma tumor micromillieu.
Macrophages are major players for both host defence as well as maintaining tissue homeostasis. In response to various tissue specific micro-environments and signals, these macrophages gets polarized to functionally distinct phenotypes like M1 and M2 with differential expression profile of cytokines, enzymes and cell surface markers (Mantovani et al 2002; Sica and Mantovani, 2012) as tabulated in Table 1. Macrophages are the most abundant immune cell population of majority of solid and hypoxic tumor micro environment, termed as tumor-associated macrophages (TAM) which, by their capacity to produce plethora of cytokines, growth factors support tumor growth, angiogenesis, and their metastasis and invasion. TAMs are also responsible for inducing endothelium anergy, thus aiding in the maintenance of tumor microenvironment while contributing for a physical barrier / challenge in majority of cancer directed immune / chemotherapies. Thus TAM poses a major hurdle in effective tumor immunotherapy and is correlated with poor prognosis in cancer patients (Sica et al 2008; Zhang et al 2010). Therefore retuning TAM to M1 phenotype represent vital and perhaps indispensible requirement of successful immunotherapy. As a new feather in the traditional radiation therapy, we have previously shown the therapeutic impact of low doses of gamma irradiation (LDI) on peritumoral macrophages as a potential non-invasive approach to improve cancer immunotherapy by enhancing tumor directed T-cell infiltration and subsequent tumor rejection (Klug and Prakash et al 2013). On the basis of these observations, we hypothesized that systemic irradiation of tumor bearing mice with LDI would be paramount for global retuning of M2 phenotype towards regulatory phenotype. In these lines, we assessed the influence of RT5 mice insulinoma microenvironment on the functional polarization of macrophages. To demonstrate the impact of low dose irradiation of M1 retuning we irradiated 25 weeks old RT5 mice systemically twice with 2Gy in neo-adjuvant and therapeutic setting and analyzed the impact of tumor irradiation of various immune related parameter by approaching 29th week demonstrated for the first time that indeed whole body irradiation of mice with LDI potentially retune M2-rich tumor microenvironment and normalizes tumor vasculature in macrophage-dependent manner.
Gamma irradiation not only recouped reduced levels of titres of M1-associated cytokines including TNF-α, IFN-γ and IL-12p70 in insulinoma but also enhanced the tumor infiltration of iNOS/CD14/CD68+ pro-inflammatory and M1 effector macrophages. This was accompanied with the tumor infiltration of T cells as well (data not shown). Most interestingly this was accompanied with reduction in the Ym-1+ TAM cells populations in the irradiated tumors, suggesting an overall M1 retuning of M2 rich tumor microenvironment of insulinoma by LDI. Such tumor irradiation of RT5 mice also resulted in higher expression of iNOS and concomitant down-regulation of M-2 associated proteins Ym-1, Fizz-1, Arginase-1, CD206 and HSP90 in both insulinoma as well purified CD11b+ peritoneal macrophages from irradiated RT mice over sham (Figure 1).
Most interestingly gamma irradiation also modulated major signalling components of these tumors as evident with increased expression of major transcription factors like pSTAT3 and to some extant pp65, which are involved in macrophage iNOS transition. On the basis of LDI mediated triggering of M1 retuning of TAM in these mice and dependency of LDI on macrophages for T cell aided immunity in these mice which we showed in our previous study, we argued logically whether M1 retuned macrophages by themselves would be important for such retuning of these tumors or not. To test our argument, macrophages were depleted from these mice by weekly application of clodronate-loaded liposomes which resulted in depletion of both pancreatic as well as intratumoral CD11b+ macrophages efficiently (Klug and Prakash et al, 2013). Since transient depletion of macrophages (week 24th through week 27th) alone in these mice could neither modify tumor micromillieu nor afforded therapeutic benefit, we depleted the macrophages at the onset of neoplasia (around week 10th through week 29th) and analyzed the impact of such long term depletion of these macrophages on tumor microenvironment and mice As expected, long term depletion of macrophages countered gamma radiation induced biochemical and immunological parameters of irradiated tumours. This was evident by complete inhibition of M1 marker like iNOS re-enhanced titre of M2 markers, immunosuppressive cytokines including VEGF, TGF-β and IL-13 and more interestingly VEGFR-2 and other angiogenesis related proteins including eNOS, Cav-1 and HSP90 also which were significantly inhibited by gamma radiation. These results altogether indicated depletion of M1 phenotype of macrophages leaving M2 rich microenvironment of these tumors which not only explained the potential reason of clodronate driven abolishment of T cell aided immunity (Klug and Prakash et al 2013) but also predisposed macrophages depleted mice for early mortality (data not shown) supporting our elucidation that presence of peritumoral iNOS+ M1 macrophages are crucial for radiation therapy.
Other then changing tumor immune parameters, gamma irradiation also targeted tumor metabolism which is important for their growth. This was evident by the rescue of blood glucose titre by gamma irradiation (Figure 1) of severely hypoglycaemic RT5 (Klug and Prakash et al, 2013). This could most likely be due to p38MAPK targeting of gamma irradiation which not only control Insulin receptor sensitivity, glucose transport but also regulate phenotype of inflammatory macrophages in both insulinoma as well macrophages (RAW264.7 and CD11b+ RT5 macrophages). This was evident by rescue of both NO production as well as induced expression of iNOS proteins in TAM in presence of p38MAPK by specific inhibitor SB203580, suggesting that p38MAPK downregulation may be involved in the regulation of iNOS expression and NO production. Thus, these results altogether demonstrated the therapeutic potential of low dose irradiation in augmenting tumor immune rejection which was guided by M1 macrophages.
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- Klug, F. et al. (2013) Low-dose irradiation programs macrophage differentiation to an iNOS⁺/M1 phenotype that orchestrates effective T cell immunotherapy. Cancer Cell, 24, 589–602.