Protocol for delivery of intraoperative immunotherapy to mice by surgical debulking of subcutaneous tumors.

Pre-clinical studies developing novel therapies to prevent cancer recurrence require appropriate surgical models. Here, we present a protocol for surgical debulking of subcutaneous tumors in mice, which allows for intraoperative application of immunotherapy-loaded biomaterials. We describe steps for inoculating tumor cells, anesthetizing mice, and performing surgery. We then detail procedures for applying biomaterial, bandaging mice, and data collection and analysis. The optimized bandaging regimen resolves the issue of wound dehiscence after surgery, for C57BL/6 mice, which interfere with surgical sites. For complete details on the use and execution of this protocol, please refer to Rwandamuriye et al.1.

A surgically optimized intraoperative poly(I:C)-releasing hydrogel prevents cancer recurrence.

Recurrences frequently occur following surgical removal of primary tumors. In many cancers, adjuvant therapies have limited efficacy. Surgery provides access to the tumor microenvironment, creating an opportunity for local therapy, in particular immunotherapy, which can induce local and systemic anti-cancer effects. Here, we develop a surgically optimized biodegradable hyaluronic acid-based hydrogel for sustained intraoperative delivery of Toll-like receptor 3 agonist poly(I:C) and demonstrate that it significantly reduces tumor recurrence after surgery in multiple mouse models. Mechanistically, poly(I:C) induces a transient interferon alpha (IFNα) response, reshaping the tumor/wound microenvironment by attracting inflammatory monocytes and depleting regulatory T cells. We demonstrate that a pre-existing IFN signature predicts response to the poly(I:C) hydrogel, which sensitizes tumors to immune checkpoint therapy. The safety, immunogenicity, and surgical feasibility are confirmed in a veterinary trial in canine soft tissue tumors. The surgically optimized poly(I:C)-loaded hydrogel provides a safe and effective approach to prevent cancer recurrence.

Temporally restricted activation of IFNß signaling underlies response to immune checkpoint therapy in mice.

The biological determinants of the response to immune checkpoint blockade (ICB) in cancer remain incompletely understood. Little is known about dynamic biological events that underpin therapeutic efficacy due to the inability to frequently sample tumours in patients. Here, we map the transcriptional profiles of 144 responding and non-responding tumours within two mouse models at four time points during ICB. We find that responding tumours display on/fast-off kinetics of type-I-interferon (IFN) signaling. Phenocopying of this kinetics using time-dependent sequential dosing of recombinant IFNs and neutralizing antibodies markedly improves ICB efficacy, but only when IFNß is targeted, not IFNα. We identify Ly6C+/CD11b+ inflammatory monocytes as the primary source of IFNß and find that active type-I-IFN signaling in tumour-infiltrating inflammatory monocytes is associated with T cell expansion in patients treated with ICB. Together, our results suggest that on/fast-off modulation of IFNß signaling is critical to the therapeutic response to ICB, which can be exploited to drive clinical outcomes towards response.

Targeting Cross-Presentation as a Route to Improve the Efficiency of Peptide-Based Cancer Vaccines.

Cross-presenting dendritic cells (DC) offer an attractive target for vaccination due to their unique ability to process exogenous antigens for presentation on MHC class I molecules. Recent reports have established that these DC express unique surface receptors and play a critical role in the initiation of anti-tumor immunity, opening the way for the development of vaccination strategies specifically targeting these cells. This study investigated whether targeting cross-presenting DC by two complementary mechanisms could improve vaccine effectiveness, in both a viral setting and in a murine melanoma model. Our novel vaccine construct contained the XCL1 ligand, to target uptake to XCR1+ cross-presenting DC, and a cell penetrating peptide (CPP) with endosomal escape properties, to enhance antigen delivery into the cross-presentation pathway. Using a prime-boost regimen, we demonstrated robust expansion of antigen-specific T cells following vaccination with our CPP-linked peptide vaccine and protective immunity against HSV-1 skin infection, where vaccine epitopes were natively expressed by the virus. Additionally, our novel vaccination strategy slowed tumor outgrowth in a B16 murine melanoma model, compared to adjuvant only controls, suggesting antigen-specific anti-tumor immunity was generated following vaccination. These findings suggest that novel strategies to target the antigen cross-presentation pathway in DC may be beneficial for the generation of anti-tumor immunity.

IFNß Is a Potent Adjuvant for Cancer Vaccination Strategies.

Cancer vaccination drives the generation of anti-tumor T cell immunity and can be enhanced by the inclusion of effective immune adjuvants such as type I interferons (IFNs). Whilst type I IFNs have been shown to promote cross-priming of T cells, the role of individual subtypes remains unclear. Here we systematically compared the capacity of distinct type I IFN subtypes to enhance T cell responses to a whole-cell vaccination strategy in a pre-clinical murine model. We show that vaccination in combination with IFNß induces significantly greater expansion of tumor-specific CD8+ T cells than the other type I IFN subtypes tested. Optimal expansion was dependent on the presence of XCR1+ dendritic cells, CD4+ T cells, and CD40/CD40L signaling. Therapeutically, vaccination with IFNß delayed tumor progression when compared to vaccination without IFN. When vaccinated in combination with anti-PD-L1 checkpoint blockade therapy (CPB), the inclusion of IFNß associated with more mice experiencing complete regression and a trend in increased overall survival. This work demonstrates the potent adjuvant activity of IFNß, highlighting its potential to enhance cancer vaccination strategies alone and in combination with CPB.

Identifying the optimal donor for natural killer cell adoptive therapy to treat paediatric B- and T-cell acute lymphoblastic leukaemia.

OBJECTIVES: Natural killer (NK) cells are an attractive source of cells for an 'off the shelf' cellular therapy because of their innate capacity to target malignant cells, and ability to be transferred between donors and patients. However, since not all NK cells are equally effective at targeting cancer, selecting the right donor for cellular therapy is critical for the success of the treatment. Recently, cellular therapies utilising NK cells from cytomegalovirus (CMV)-seropositive donors have been explored. However, whether these NK cells are the best source to treat paediatric acute lymphoblastic leukaemia (ALL) remains unclear. METHODS: Using a panel of patient-derived paediatric B- and T-ALL, we assessed the ability of NK cells from 49 healthy donors to mount an effective functional response against these two major subtypes of ALL. RESULTS: From this cohort, we have identified a pool of donors with superior activity against multiple ALL cells. While these donors were more likely to be CMV+, we identified multiple CMVneg donors within this group. Furthermore, NK cells from these donors recognised B- and T-ALL through different activating receptors. Dividing functional NK cells into 29 unique subsets, we observed that within each individual the same NK cell subsets dominated across all ALL cells. Intriguingly, this occurred despite the ALL cells in our panel expressing different combinations of NK cell ligands. Finally, we can demonstrate that cellular therapy products derived from these superior donors significantly delayed leukaemia progression in preclinical models of ALL. CONCLUSIONS: We have identified a pool of superior donors that are effective against a range of ALL cells, representing a potential pool of donors that can be used as an adoptive NK cell therapy to treat paediatric ALL.

Diverse Anti-Tumor Immune Potential Driven by Individual IFNα Subtypes.

Immunotherapies harnessing T cell immunity have shown remarkable clinical success for the management of cancer. However, only a proportion of patients benefit from these treatments. The presence of type I interferon (IFN) within the tumor microenvironment is critical for driving effective tumor-specific T cell immunity. Individuals can produce 12 distinct subtypes of IFNα, which all signal through a common receptor. Despite reported differences in anti-viral potencies, the concept that distinct IFNα subtypes can improve anti-cancer treatments remains unclear. We tested whether expression of unique IFNα subtypes confined to the tumor microenvironment enhances tumor control. This was systematically evaluated by transplantation of B16 murine melanoma cells secreting five unique IFNα subtypes (B16_IFNα2; B16_IFNα4; B16_IFNα5; B16_IFNα6; B16_IFNα9) into a pre-clinical murine model. We show that IFNα2 and IFNα9 are the only subtypes capable of completely controlling tumor outgrowth, with this protection dependent on the presence of an adaptive immune response. We next determined whether these differences extended to other model systems and found that the adoptive transfer of tumor-specific CD8+ T cells engineered to secrete IFNα9 delays tumor growth significantly and improves survival, whereas no enhanced survival was observed using T cells secreting IFNα4. Overall, our data shows that the expression of distinct IFNα subtypes within the tumor microenvironment results in different anti-tumor activities, and differentially affects the efficacy of a cancer therapy targeting established disease.

Impaired T cell proliferation by ex vivo BET-inhibition impedes adoptive immunotherapy in a murine melanoma model.

Activation of naïve CD8+ T cells stimulates proliferation and differentiation into cytotoxic T-lymphocytes (CTLs). Adoptive T Cell Therapy (ACT) involves multiple rounds of ex vivo activation to generate enough CTLs for reinfusion into patients, but this drives differentiation into terminal effector T cells. Less differentiated CTL populations, such as stem cell memory T cells, are more ideal candidates for ACT because of increased self-renewal and persistent properties. Ex vivo targeting of T cell differentiation with epigenetic modifiers is a potential strategy to improve cytotoxic T-lymphocyte (CTL) generation for ACT. We established a pipeline to assess the effects of epigenetic modifiers on CD8+ T cell proliferation, differentiation, and efficacy in a preclinical melanoma model. Single treatment with epigenetic modifiers inhibited T cell proliferation in vitro, producing CD44hiCD62Lhi effector-like T cells rather than a stem cell memory T cell phenotype. Most epigenetic modifying agents had no significant effect on ACT efficacy with the notable exception of the bromodomain and extraterminal (BET)-inhibitor JQ1 which was associated with a decrease in efficacy compared to unmodified T cells. These findings reveal the complexity of epigenetic targeting of T cell differentiation, highlighting the need to precisely define the epigenetic targeting strategies to improve CTL generation for ACT.

Acquired resistance during adoptive cell therapy by transcriptional silencing of immunogenic antigens.

Immunotherapies such as adoptive cell therapy (ACT) are promising treatments for solid cancers. However, relapsing disease remains a problem and the molecular mechanisms underlying resistance are poorly defined. We postulated that the deregulated epigenetic landscape in cancer cells could underpin the acquisition of resistance to immunotherapy. To address this question, two preclinical models of ACT were employed to study transcriptional and epigenetic regulatory processes within ACT-treated cancer cells. In these models ACT consistently causes robust tumor regression, but resistance develops and tumors relapse. We identified down-regulated expression of immunogenic antigens at the mRNA level correlated with escape from immune control. To determine whether this down-regulation was under epigenetic control, we treated escaped tumor cells with DNA demethylating agents, azacytidine (AZA) and decitabine (DEC). AZA or DEC treatment restored antigen expression in a proportion of the tumor population. To explore the importance of other epigenetic modifications we isolated tumor cells refractory to DNA demethylation and screened clones against a panel of 19 different epigenetic modifying agents (EMAs). The library of EMAs included inhibitors of a range of chromosomal and transcription regulatory protein complexes, however, when tested as single agents none restored further antigen expression. These findings suggest that tumor cells employ multiple epigenetic and genetic mechanisms to evade immune control, and a combinatorial approach employing several EMAs targeting transcription and genome stability may be required to overcome tumor resistance to immunotherapy.

Dendritic Cells and Cancer: From Biology to Therapeutic Intervention.

Inducing effective anti-tumor immunity has become a major therapeutic strategy against cancer. Dendritic cells (DC) are a heterogenous population of antigen presenting cells that infiltrate tumors. While DC play a critical role in the priming and maintenance of local immunity, their functions are often diminished, or suppressed, by factors encountered in the tumor microenvironment. Furthermore, DC populations with immunosuppressive activities are also recruited to tumors, limiting T cell infiltration and promoting tumor growth. Anti-cancer therapies can impact the function of tumor-associated DC and/or alter their phenotype. Therefore, the design of effective anti-cancer therapies for clinical translation should consider how best to boost tumor-associated DC function to drive anti-tumor immunity. In this review, we discuss the different subsets of tumor-infiltrating DC and their role in anti-tumor immunity. Moreover, we describe strategies to enhance DC function within tumors and harness these cells for effective tumor immunotherapy.

CD8+XCR1neg Dendritic Cells Express High Levels of Toll-Like Receptor 5 and a Unique Complement of Endocytic Receptors.

Conventional dendritic cells (cDC) resident in the lymphoid organs of mice have been classically divided into CD8+ and CD8neg subsets. It is well-established that CD8+ dendritic cells (DCs) and their migratory counterparts in the periphery comprise the cross-presenting cDC1 subset. In contrast, CD8neg DCs are grouped together in the heterogeneous cDC2 subset. CD8neg DCs are relatively poor cross-presenters and drive more prominent CD4+ T cell responses against exogenous antigens. The discovery of the X-C motif chemokine receptor 1 (XCR1) as a specific marker of cross-presenting DCs, has led to the identification of a divergent subset of CD8+ DCs that lacks the ability to cross-present. Here, we report that these poorly characterized CD8+XCR1neg DCs have a gene expression profile that is consistent with both plasmacytoid DCs (pDCs) and cDC2. Our data demonstrate that CD8+XCR1neg DCs possess a unique pattern of endocytic receptors and a restricted toll-like receptor (TLR) profile that is particularly enriched for TLR5, giving them a unique position within the DC immunosurveillance network.

Cross-presentation of cutaneous melanoma antigen by migratory XCR1+CD103- and XCR1+CD103+ dendritic cells.

The question of which dendritic cells (DCs) cross-present peripheral tumor antigens remains unanswered. We assessed the ability of multiple skin-derived and lymphoid resident DCs to perform this function in a novel orthotopic murine melanoma model where tumor establishment and expansion is within the skin. Two migratory populations defined as CD103-XCR1+ and CD103+XCR1+ efficiently cross-presented melanoma-derived antigen, with the CD103-XCR1+ DCs surprisingly dominating this process. These results are critical for understanding how antitumor CD8+ T cell immunity is coordinated to tumor antigens present within the skin.

The efficacy of tumor debulking surgery is improved by adjuvant immunotherapy using imiquimod and anti-CD40.

BACKGROUND: Tumor debulking surgery followed by adjuvant chemotherapy or radiotherapy is a standard treatment for many solid malignancies. Although this approach can be effective, it often has limited success against recurrent or metastatic cancers and new multimodality approaches are needed. Adjuvant immunotherapy is another potentially effective approach. We therefore tested the efficacy of the TLR7 agonist imiquimod (IMQ) combined with agonistic anti-CD40 in an incomplete debulking model of malignant mesothelioma. METHODS: Established subcutaneous murine ABA-HA mesothelioma tumors in BALB/c mice were surgically debulked by 75% and treated with either: i) saline; ii) intratumoral IMQ; iii) systemic anti-CD40 antibody, or using a combination of IMQ and anti-CD40. Tumour growth and survival were monitored, and the role of anti-tumor CD4 and CD8 T cells in therapeutic responses was determined. RESULTS: The combination therapy of partial debulking surgery, IMQ and anti-CD40 significantly delayed tumor growth in a CD8 T cell dependent manner, and promoted tumor regression in 25% of animals with establishment of immunological memory. This response was associated with an increase in ICOS+ CD8 T cells and tumor-specific CTL activity in tumor draining lymph nodes along with an increase in ICOS+ CD8 T cells in responding tumours. CONCLUSIONS: We show that the post-surgical environment can be significantly altered by the co-administration of adjuvant IMQ and anti-CD40, resulting in strong, systemic anti-tumor activity. Both adjuvants are available for clinical use/trial, hence this treatment regimen has clear translational potential.

Neoadjuvant anti-tumor vaccination prior to surgery enhances survival.

BACKGROUND: This study was conducted to determine if anti-tumor vaccination administered prior to partial debulking surgery could improve survival using a murine solid tumour model. METHODS: Tumor incidence and survival rates were compared in mice bearing subcutaneous AB1-HA mesothelioma tumors that received either sham surgery, debulking surgery or vaccination prior to debulking surgery. Additionally, mice were depleted of CD4 and/or CD8 T lymphocytes during vaccination to assess their involvement in vaccine induced anti-tumor immunity. Flow cytometry was performed to characterise changes in the proportion and activation status of immune cells associated with anti-tumor immunity. RESULTS: Neoadjuvant vaccination combined with debulking surgery resulted in decreased tumor burden, increased survival and generation of tumor-specific immunity compared to surgery alone. Depletion of CD8 T cells completely abrogated any vaccine induced anti-tumor immune response. Conversely, CD4 depletion enhanced CD8 T cell activation resulting in complete tumor regression in 70% of mice treated with combined surgery and vaccination therapy. Tumor free survival was associated with established immunological memory as defined by the induction of effector memory T cells and resistance to rechallenge with parental AB1 mesothelioma cells. CONCLUSIONS: Neoadjuvant anti-cancer vaccination combined with partial debulking surgery induced CD8-dependent anti-tumor immunity that significantly delayed tumor outgrowth relative to surgery alone. Complete tumor eradication was observed when vaccination and surgery were performed in CD4 T cell depleted animals. This demonstrates that adjuvant immunotherapy can improve post-surgical survival following cancer debulking surgery and provides a scientific rational for clinical trials of such an approach.

Long-term exposure of mesothelial cells to SV40 and asbestos leads to malignant transformation and chemotherapy resistance.

Simian virus 40 (SV40) has been implicated in the development of several cancers including malignant mesothelioma. A definitive role for the virus in human mesothelioma has not been unequivocally demonstrated but has been rigorously debated. The virus clearly has oncogenic potential: the TAg is one of the most potent transforming proteins known and acts synergistically with crocidolite asbestos to transform mesothelial cells. In this study, we show that SV40 oncogenes alone can cause malignant transformation and that asbestos-induced DNA damage and apoptosis occurs principally in cycling cells. After long-term exposure (up to 100 days) to both SV40 and asbestos, cells become resistant to stress-induced senescence. Significantly, these cells demonstrate resistance to chemotherapy-induced apoptosis. This finding has implications for the development of effective treatment options for patients with mesothelioma.

Resident CD8(+) and migratory CD103(+) dendritic cells control CD8 T cell immunity during acute influenza infection.

The identification of the specific DC subsets providing a critical role in presenting influenza antigens to naïve T cell precursors remains contentious and under considerable debate. Here we show that CD8(+) T lymphocyte (TCD8+) responses are severely hampered in C57BL/6 mice deficient in the transcription factor Batf3 after intranasal challenge with influenza A virus (IAV). This transcription factor is required for the development of lymph node resident CD8(+) and migratory CD103(+)CD11b(-) DCs and we found both of these subtypes could efficiently stimulate anti-IAV TCD8+. Using a similar ex vivo approach, many publications on this subject matter excluded a role for resident, non-migratory CD8(+) DC. We postulate the differences reported can partially be explained by how DC are phenotyped, namely the use of MHC class II to segregate subtypes. Our results show that resident CD8(+) DC upregulate this marker during IAV infection and we advise against its use when isolating DC subtypes.

Tumor cells, rather than dendritic cells, deliver antigen to the lymph node for cross-presentation.

It is widely accepted that generation of tumor specific CD8+ T-cell responses occur via cross-priming; however the source of tumor antigen for this event is unknown. We examined the source and form of tumor antigen required for cross-presentation in the local lymph node (LN) using a syngeneic mouse tumor model expressing a marker antigen. We found that cross-presentation of this model tumor antigen in the LN is dependent on continuous traffic of antigen from the tumor site, but without any detectable migration of tumor resident dendritic cells (DCs). Instead, small numbers of tumor cells metastasize to local LNs where they are exposed to a localized CTL attack, resulting in delivery of tumor antigen into the cross-presentation pathway.