AuthOr’s VIew OncoImmunology 1:9, 1626–1628; December 2012; © 2012 Landes Bioscience Chemoimmunotherapy for advanced gastrointestinal carcinomas A successful combination of gene therapy and cyclophosphamide Mariana Malvicini,1 Flavia Piccioni,1 Juan Bayo,1 esteban Fiore,1 Catalina Atorrasagasti,1 Laura Alaniz,1 Mariana García,1 Jorge B. Aquino,1 Manuel Gidekel,4 Pablo Matar2,3 and Guillermo Mazzolini1,3,* 1Gene therapy Laboratory; Liver unit; school of Medicine; Austral university; Buenos Aires, Argentina; 2Institute of experimental Genetics; school of Medical sciences; National university of rosario; rosario, Argentina; 3CONICet (Consejo Nacional de Investigaciones Científicas y técnicas); Buenos Aires, Argentina; 4universidad de la Frontera; temuco, Chile Keywords: advanced gastrointestinal carcinomas, cyclophosphamide, gene therapy, immunomodulation, interleukin-12 the combination of a single low dose of cyclophosphamide (Cy) with the adenovirus-mediated gene transfer of interleukin-12 (AdIL-12) might represent a successful therapy for experimental gastrointestinal tumors. this approach has been proven to revert immunosuppressive mechanisms elicited by cancer cells and to synergistically promote antitumor immunity. In addition, this therapeutic regimen has been shown to be more efficient in achieving complete tumor regressions in mice than the application of a metronomic schedule of Cy plus AdIL-12. During the last two decades, a number macrophage colony-stimulating factor the immunosuppressive activity Tregs, of immunotherapy-based strategies for (GM-CSF) and tumor necrosis factor α including secretion of specific cytokines advanced gastrointestinal carcinomas (TNFα) has been shown to be efficient and the induction of apoptosis in effec- (GICs) has given promising results, not in generating immunity against many tor T cells.6 We found that the combina- only at preclinical stage but also in the tumors, although it is frequently associ- tion of Cy Mo and AdIL-12 significantly clinic.1 However, immunotherapeutic ated with toxicity.4 We have recently dem- reduced the production of IL-10 and approaches have to face an important onstrated that a combination of IL-12, a transforming growth factor β (TGFβ) obstacle: the ability of tumor cells to evade potent antitumor cytokine and a single by Tregs and to abolish their capacity to immune attack.2 Several immunosuppres- low dose of cyclophosphamide (Cy Mo) inhibit lymphocyte proliferation. Thus, sive mechanisms elicited by cancer cells synergize with sub-therapeutic doses of an the depletion and/or inhibition of Tregs have been identified in animal models and adenovirus expressing IL-12 (AdIL-12) to function appears to be the main immu- in patients including:1 loss of MHC Class I generate an immune response that is able noregulatory mechanism associated with molecules from the surface of tumor cells,2 to eradicate established tumor nodules. the antitumor effects of Cy Mo and increased oxidative stress and3 recruit- Such a combination regimen was found AdIL-12.5 Myeloid cells with immuno- ment of myeloid-derived suppressor cells to mediate powerful antitumor effects suppressive functions, including MDSCs (MDSCs) and regulatory T cells (Tregs). against subcutaneous colorectal carci- or tumor-associated macrophages A correlation between increased levels of noma (CRC), metastatic CRC and pan- (TAM), have recently received much these immunosuppressive cell populations creatic cancer in mice.5 attention in the field of tumor immunol- and poor prognosis has been observed in The combination of Cy Mo and ogy, as they emerged as potent suppres- many types of cancer.3 AdIL-12 seems to have a great impact sors of T cell-mediated immunity with a Increasing evidence suggests that on the immunosuppressive tumor micro- wide repertoire of effector mechanisms.7 immune responses are involved in the con- environment, resulting in the deple- The combination of Cy Mo and AdIL-12 trol of cancer and that the immune system tion of Tregs. Consistently, the adoptive also reduced the number of MDSCs in can be manipulated in different ways to transfer of Tregs significantly abolished the spleen of tumor-bearing mice, hence recognize and fight cancer cells. The sys- the antitumoral effects achieved by representing a valuable strategy to impact temic administration of cytokines such as Cy Mo and AdIL-12. Several mecha- on cell populations critically involved in interleukin (IL)-12, IL-15, granulocyte nisms have been proposed to explain tumor-induced immunosuppression. *Correspondence to: Guillermo Mazzolini; Email: [email protected] Submitted: 07/21/12; Accepted: 07/26/12 http://dx.doi.org/10.4161/onci.21651 1626 OncoImmunology Volume 1 Issue 9 AuthOr’s VIew AuthOr’s VIew Successful immunotherapies not only reverse immunosuppressive mechanisms elicited along with tumor progression but also induce a strong cytotoxic immune response against cancer cells. The induc- tion of antitumor immunity requires the active participation of antigen-presenting cells such as dendritic cells (DCs), which are essential for the optimal activation of T-cell responses against cancer. The use of Cy Mo contributed to increase tumor immunogenicity and to the generation of specific CTL responses by altering the maturation and/or functional status of DCs. This activity has been shown to potentiate the efficacy of gene therapy with AdIL-12.8 In addition, Cy Mo plus AdIL-12 induces the expansion of inter- feron γ (IFNγ)-secreting CD4+ T lym- phocytes with specific cytotoxic activity against CRC cells5 and promotes a strong tumor infiltration by CD4+ and CD8+ T cells (Fig. 1). Previous studies have shown that Cy might potentiate the antitumor efficacy of a number of immunotherapeutic strat- egies. The immunomodulatory effects of Cy include a Th2/Th1 cytokine shift, the stimulation of innate immune responses, the activation of DCs and the elimination or inhibition of Tregs. Although the mechanisms of action of chemotherapeutic agents are not fully understood, it has recently been dem- onstrated that autophagy in cancer cells is necessary for immunogenic cell death and hence underlies optimal therapeutic effects.9 Our in vivo models suggest that some of these mechanisms are involved in the antitumor effects of Cy Mo plus Figure 1. Proposed mechanisms of action underlying tumor regression as induced by the com- bination of monodose cyclophosphamide (Cy Mo) and IL12 gene transfer. Factors secreted by the AdIL-12. tumor induce the mobilization of cells with immunosuppressive activity such as tregs, myeloid- The application of a Cy metronomic derived suppressor cells (MDsCs) and tolerogenic dendritic cells (iDCs). these cells can kill tumor- schedule (frequent and homogeneously specific t cells or inhibit their activity by inducing a state of anergy. the sequential administration spaced low-dose administrations) has of single dose (Cy Mo) or metronomic dose of Cy (Cy Me) followed by IL12 gene transfer (AdIL-12) been shown to induce immunostimula- releases these brakes. however, after several weeks of treatment, Cy Me plus AdIL-12 is not as ef- ficient as Cy Mo plus AdIL-12 in maintaining MDsCs to low levels. On the other hand, Cy Mo might tory and antiangiogenic effects, opening promote the immunogenicity of cancer cells by improving the activation and/or maturation of up new avenues for cancer immunother- antigen presenting cells. In addition, Cy Mo plus AdIL-12 stimulate the infiltration of CD4+ and apy combinations.10 We therefore tested CD8+ t lymphocytes while Cy Me fails not only to generate a beneficial tumor microenvironment metronomic Cy (Cy Me) schemes, looking but also to induce tumor-specific immune responses. for a possible boosting in the efficacy of AdIL-12. However, this protocol was less to maintain such an inhibition over time. DCs and to generate a strong and specific effective than that with the combination of Therefore, the combination of Cy Me and response against CRC cells (Fig. 1). These Cy Mo and AdIL-12. Indeed, although Cy AdIL-12 was not as efficient as that of Cy observations might have significant impli- Me plus AdIL-12 was able to decrease the Mo and AdIL-12 in eliminating MDSCs. cations for future strategies based on the incidence of Tregs and (initially) MDSCs In addition, this therapeutic approach use of Cy as an immunomodulatory adju- similar to Cy Mo plus AdIL-12, it failed failed to induce a powerful maturation of vant including DC-based vaccines. www.landesbioscience.com OncoImmunology 1627 The possibility of manipulating the antitumor responses and inhibit immuno- AdIL-12 perhaps represents a good strategy immune system against cancer would subversion mechanisms elicited by cancer to improve the immune recognition of tumor be absolutely thrilling, and combination cells could be of paramount importance. In cells and, hence, to achieve an optimal thera- approaches that both stimulate potent this regard, the combination of Cy Mo and peutic response that eradicates GICs. 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