Combined mitoxantrone and anti-TGFβ treatment with PD-1 blockade enhances antitumor immunity by remodelling the tumor immune landscape in neuroblastoma

Immune checkpoint inhibitors (ICI) have shown impressive clinical results against a variety of highly aggressive tumors, although durable benefits have only been observed in a limited fraction of patients [1‐3]. The main reason for this non-response is the lack of functional tumor-infiltrating T cells [4]. Given the great potential of ICI, it is of paramount importance to improve their efficacy by populating less infiltrated tumors with functional immune cells.

Compelling evidence shows that in order to function, T cells required a favourable immune microenvironment in which other immune actors play a crucial role [5]. Indeed, tumors that respond to immunotherapy are enriched not only in T cells, but also in other immune cell populations, including conventional type 1 dendritic cells (DC1) and natural killer (NK) cells, which, by interacting with each other, create an environment suitable for the priming and expansion of tumor-specific T cells [5]. Consistently, similarly to CD8⁺ T cells, intratumoral DC1 and NK cells have also been associated with a strong antitumor response and favourable clinical outcome in various tumors [6]. The development of novel approaches that can restore the function of the entire immune cycle is therefore of crucial importance to increase the number of patients who can benefit from immunotherapy. In this regard, several chemotherapeutic treatments have been shown to recall immune cells in the tumor microenvironment (TME) through different mechanisms [7]. For examples, some chemotherapeutics such as doxorubicin (DX), mitoxantrone (MTX), oxaliplatin (OXP), and cyclophosphamide, when administrated at low doses are able to induce immunogenic cell death (ICD) by promoting DC activation, antigen presentation and priming of tumor-specific CD8⁺ T cells [7‐10].

However, an increase in intratumoral immune cell infiltrate may not be conclusive because of the multiple mechanisms adopted by tumors to prevent patients’ immune system from targeting and eliminating their own tumor cells [3]. Recently, transcriptional profiling of melanoma and metastatic urothelial carcinoma patients unresponsive to anti-PD-1 therapy revealed an enrichment in pathways associated to TGFβ [11, 12], a cytokine produced by the TME that reduces immune cell recall within the tumor and inhibits the function of both T cells and NK cells [13]. Consistently, recent use of TGFβ-blocking antibodies has been shown to overcome resistance to anti-PD-1 therapy by reactivating the antitumor immune response in colorectal cancer and melanomas [12, 14].

Approaches aimed at improving the effectiveness of treatments in a increasing number of patients, in combination with therapies that enhance the recruitment of innate and adaptive immune cells, could prove valuable for currently intractable tumors, such as neuroblastoma (NB), a paediatric cancer of the peripheral sympathetic system that accounts for 15% of all childhood cancer-related deaths [15‐19]. Despite advanced and intensive multidisciplinary therapeutic approaches based on induction chemotherapy, myeloablative chemotherapy, surgery, radiotherapy and treatment of minimal residual disease, mortality of patients with high-risk NB remains significant, and those who survive experience severe long-term side effects. Therefore, it is important to develop more effective and tolerated therapeutic approaches.

Extensive pathological studies of over than 100 cases of primary and metastatic human NBs examined by immunohistochemistry (IHC) revealed the importance of the immune content in the NB microenvironment [20‐22]. NB highly infiltrated by T cells are also enriched with DC and NK cells, and the abundance of these immune cell populations is positively correlated with a favourable clinical outcome [20‐22]. The importance of the intratumoral immune context in NB is further strengthened by the identification of intratumoral gene signatures specific to these immune cell populations that correlate with PD-1 and PD-L1 expression levels [22].

Similarly to other tumors, NB exploits a variety of immune evasion strategies including expression of immune checkpoint molecules, induction of immunosuppressive cells, as well as secretion of immunomodulatory mediators, including TGFβ [23‐26]. The development of novel multiple combined immunotherapeutic protocols to recruit innate and adaptive immune cells and convert the immunosuppressive environment to an immunostimulatory one may be effective in the treatment of NB.

In this work, we employ a combinatorial in situ immunomodulation strategy based on the administration of i) low dose of chemotherapy to promote ICD and mobilize immune cells into the TME, ii) anti-TGFβ to overcome immunosuppression in the TME, iii) and anti-PD-1 to restore the function of tumor-infiltrating immune cells. Using ex vivo approaches with murine- and patients-derived NB tissues, as well mouse models of NB grown subcutaneously or orthotopically in the adrenal gland, we identify a novel combination therapy that recruits a variety of innate and adaptive immune cells into the TME, and significantly reduces the growth of this aggressive tumor.

This study demonstrates that low-dose MTX curbs in vivo NB growth leading to substantial tumor regression and remodeling of the tumor’s immune landscape when combined with PD-1 and TGFβ blockade. This combined treatment is also able of inducing the recruitment of immune cells and the activation of CD8⁺ T cells and NK cells against PDOTS generated from human NB patients.

ICIs have been extensively shown to trigger T-cell effector function to control tumor growth in both mice and human cancers [46, 47]. However, antitumor immune responses to individual immunotherapeutic agents remain limited to a subset of patients [1]. This is because immunotherapy is ineffective for tumors that, as high-risk NB, are both lacking immune cells and characterized by an immunosuppressive TME [1]. Therefore, developing combination therapies to induce recruitment of immune cells and target the immunosuppressive factors released in the TME could be a successful strategy to improve the efficacy of treatment regimens [48].

Chemotherapy agents, originally known to directly inhibit or kill malignant cells, have recently been found to promote antitumor immunity by increasing tumor immunogenicity, enhancing T-cell infiltration, or reducing immunosuppressive cell populations [49]. Some of these chemotherapy drugs are known to activate tumor-specific T cells by inducing ICD when administered at low doses [7]. The effectiveness of these drugs increases in association with immune checkpoint therapies [50]. Indeed, combined treatment of DX with PD-1 or PD-L1 antibodies resulted in a significant increase in efficacy in metastatic triple-negative human breast cancer and in various murine tumors, such as melanoma and breast cancer [51, 52]. Similarly, OXP treatment has been found to increase the efficacy of anti-PD-L1 therapy in murine colorectal cancer [53].

NB cells are capable of secreting a variety of soluble mediators that can suppress lymphocyte activation, including TGFβ [23, 38, 54]. Indeed, high levels of tumor TGFβ have been associated with reduced event free survival [43]. Activation of the TGFβ pathway has also recently emerged as a potential factor responsible for primary resistance to immune checkpoint blockade therapy [11, 12]. Combined anti-TGFβ/anti-PD-1 treatment led to profound and durable antitumor responses in urothelial, melanoma, and breast cancer models, and promoted the establishment of immunological memory in a tumor rechallenge model [55, 56]. The induction of long-term tumor-specific T-cell memory is attributed to immune checkpoint PD-1 and PD-Ll blockade [55, 56]. In this context, TGFβ blockade has been found to restore an immunity-friendly environment in NB capable of unleashing the full potential of reactive immune cells and increasing their persistence [57]. Based on these considerations, here we evaluated the immunomodulatory effect of 6 chemotherapy agents administered at low doses, including those used in the therapy of high-risk NB and/or able to induce ICD, in two transplantable 9464D and 975A2 murine models that closely recapitulate the molecular and biological features of high-risk human NBs [27, 58‐61]. Low-dose MTX was found to have the greatest immunostimulatory capacity able to attract immune cells into the NB microenvironment in ex-vivo and in vivo approaches. To date, MTX has been approved for the treatment of several tumors, including acute non-lymphoblastic leukemia and some advanced forms of prostate cancer (clinicaltrials.gov). We found that low-dose MTX triggers T-cell priming in NB through induction of ICD [62], making the tumor more susceptible to immune-mediated attack by up-regulating the expression of MHC class I molecules (Supplementary Fig. S12). Interestingly, low-dose MTX also results in a concomitant increase in PD-L1 levels (Supplementary Fig. S12), thus strengthening the idea that chemotherapy alone may not provide a lasting therapeutic effect. This finding is consistent with the recent evidence indicating that treatment regimens combining multiple immunotherapeutic strategies, some of which even engaging innate and adaptive immunity, are more effective in NB patients than monotherapies [63‐65]. We found that low-dose MTX in combination with TGFβ and PD-1 blockers is able to remodeling the landscape of tumor-infiltrating immune cells by compensating for the lack of immune cells that characterize high-risk NBs [20‐22] (Fig. 7F). Consistently, dual PD-1/TGFβ blockade has recently been shown to i) make human tumor cells more sensitive to different chemotherapeutic agents by altering their plasticity [66]; ii) enhance the cytolytic activity of NK and T cells towards tumor cells [67‐69]; and iii) up-regulate the expression of immune response genes, including those encoding multiple chemokines, such as CCL5, associated with immune cell infiltration and enhanced anti-tumor activity [70]. Indeed, MTP treatment resulted in an enrichment of DCs and activated CD8⁺ T cells and NK cells in both mouse models of NB, supporting the key role of these immune cells in controlling NB growth, as observed in human NB speciments [20, 22]. We noted that this treatment also induced an enrichment of immunosuppressive populations, such as Treg and neutrophils. The increase of Tregs is unexpected considering the role of TGFβ in maintaining peripheral Treg cells [71]. We interpret this increase as indicative of a strong immune response that results in the recruitment of immune cells, including Treg, to sites of inflammation. As seen for other tumors, the presence of activated effector cells is indicative that the increase in Treg is not sufficient to create a substantially immunosuppressive TME [39]. Like macrophages, murine neutrophils are distinguished into antitumor and protumor neutrophils [72‐74]. In the early phase of tumorigenesis, neutrophils appear to contribute to the antitumor immune response, perhaps through stimulation of adaptive immunity and activation of CD8⁺ T cells [72]. Cross-talk between neutrophils and activated T cells resulted in substantial upregulation of the costimulatory molecules on the surface of neutrophils, capable of enhancing T-cell proliferation in a positive feedback loop [75].

Recent evidence highlighted that, even if the adaptive immune system is compromised [76], or T-cell function cannot be fully recovered by PD-1 inhibitors under specific circumstances [77], PD-1/PD-L1 inhibitors can still increase antitumor efficacy. This is because other types of immune cells, such as DCs, TAMs and NK cells, are also responsive to PD-1/PD-L1 antagonists [76, 78‐82], thus strengthening the use of these agents to increase antitumor efficacy.

In this regard, we can assume that the addition of anti-TGFβ along with checkpoint blockade interrupts a hierarchy of immunosuppressive events, consisting of TGFβ that dampens the initial immune response by preventing immune cells from infiltrating tumors, and PD-1/PD-L1 signaling that operating at a later stage suppresses the effector functions of immune cells causing their depletion [83].

Immune checkpoint blockade is known to reactivate pre-existing intratumoral T cells in human cancer lesions. This reactivation is accompanied by an increase in the chemoattractant production by different immune cell populations [4, 46, 84]. Interestingly, we noted that, compared with MTX alone, treatment with MTP resulted in a simultaneously increase of different chemokines involved in the recruitment of both lymphoid and myeloid cell populations (Fig. 6G). In addition to promoting recruitment and effector function of tumor-specific CD8⁺ T cells, these chemokines are also able to enrich the TME of functional DC and NK cells, two key immune components associated with improved survival of both adult and pediatric cancer patients [5]. Particularly important is the increase of CCL5, produced by CD8⁺ T cells, NK cells and innate lymphoid cells, that is crucial for recruitment of cDC1s, macrophages and Treg cells in the TME [85]; CXCL9, CXCL10 and CXCL16, produced by DCs and macrophages, that induce the recruitment and activation of NK cells, NKT cells and CD8⁺ T cells [5, 86‐88]; CCL21, that significantly increase the proportion of T cells, NK cells and DCs within the tumor [89]; CD40 and FLT3L, produced by intratumoral NK cells, which supports the viability and functions of cDC1s within the TME by promoting their local differentiation from precursor cells [19, 90]. Interestingly, CXCL9 expression by cDCs has been previously described to mediate the clustering of DC-T cells within lymph nodes [91], and since interaction between these two immune cell populations is quite rare in tumors [22, 92], it is possible that increased CXCL9 expression by facilitating these interactions may promote T-cell effector function.

Finally, our findings in human NB specimens strengthen the evidence found in mouse models, providing proof of principle that the proposed combinatorial strategy may serve as a potential alternative therapy in the NB clinic. So far, the treatment protocols under investigation have mostly been derived from therapeutic regimens formulated for adult tumors [93]. This is a major limitation because childhood tumors are genetically different from their adult counterparts [94], suggesting the need for alternative therapeutic approaches. Up to now, few studies have combined immunotherapy and chemotherapy in pediatric cancers, mainly due to the original idea that chemotherapy being immunosuppressive may act by inhibiting the beneficts achieved with immunotherapy [95]. Instead, we believe that the use of metronomic low dose of chemotherapeutics such as MTX, capable of exposing the host immune system to large amounts of tumor antigens and damage-associate molecular patterns (DAMPs), could overcome the relative coldness of childhood cancer, as high-risk NB. Therefore, this study provides a rational approach based on intra-tumoral recall of immune effector cells that allows for greater efficacy of the proposed immunotherapy [96, 97]. Further investigation in a prospective study with a larger number of human NB samples, will be crucial to confirm therapeutic efficacy of MTP treatment.

The results of this study show that low-dose MTX in combination with TGFβ and PD-1 blockade remodels intratumoral immune cell type and density and control tumor growth, thus suggesting its use in patients with poorly infiltrated T-cell tumors refractory to immunotherapy, such as high-risk NBs.