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Nanoparticle-Mediated Remodeling of the Tumor Microenvironment to Enhance Immunotherapy Sara Musetti, and Leaf Huang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b05893 • Publication Date (Web): 03 Dec 2018 Downloaded from http://pubs.acs.org on December 3, 2018

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Nanoparticle-Mediated Remodeling of the Tumor Microenvironment to Enhance Immunotherapy Sara Musetti and Leaf Huang* Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 *corresponding author: [email protected]

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Abstract Nanoscience has long been lauded as a method through which to overcome tumor-associated barriers. As successful as cancer immunotherapy has been, limitations associated with the tumor microenvironment or side effects of systemic treatment have become more apparent. In this review, we seek to lay out the therapeutic challenges associated with the tumor microenvironment and the ways in which nanoscience is being applied to remodel the tumor microenvironment and increase the susceptibility of many cancer types to immunotherapy. We detail the nanomedicines on the cutting edge of cancer immunotherapy and how their interactions with the TME make them more effective than systemically administered immunotherapies.

Keywords: nanoparticles, cancer, immunotherapy, tumor microenvironment, nanovaccine, cytokines, checkpoint inhibitors, immunogenic cell death, cytotoxic T cells


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Vocabulary: Tumor microenvironment: The complex system of immune cells, structural cells, cytokines, and extracellular matrix that surround and support tumor nests, creating a barrier to therapy. EPR Effect: Enhanced permeation and retention effect created by leaky, abnormal vasculature that supports the uptake of nanomaterials selectively into the tumor over other organs. Cytotoxic T cells: T cells that recognize specific threats, including cancer cells, and selectively kill them. Nanovaccine: Nanoparticles used to stimulate immune responses against a specific antigen or set of antigens, often by delivering adjuvants and antigens directly to antigen-presenting cells. Immunogenic cell death: A specialized form of cell death induced by low doses of chemotherapeutics that causes tumor cells to release antigens, danger-associated molecular patterns, and other molecular signals that stimulate the immune system. Checkpoint inhibitors: Molecules that block immunosuppressive checkpoint receptors on immune cells or tumor cells that prevent the immune system from raising a robust anticancer response.


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One of the fundamental mischaracterizations of tumors is the mental image of them as a simple mass of transformed cells, growing uncontrollably. While cancerous cells do exhibit these growth characteristics, tumors themselves are far more complex. Tumors and their microenvironments are elaborate conglomerations of transformed cells, blood vessels, fibroblasts, and immune cells, all producing a soup of cytokines that enhance tumor cell growth and suppress anti-tumor immune activation. Cancer cells are often so heavily mutated that the immune system could recognize them as potential targets; however, the immunosuppressive nature of the tumor microenvironment inhibits the immune system’s ability to do its job. Over the past decade or so, cancer immunology has grown as a field as scientists and clinicians have been forced to reckon with the fact that inhibiting cancer cells alone simply is not enough to effectively treat cancer. Nanoscience as a means of targeted cancer therapy has been growing in tandem with cancer immunology, and as each field advanced, they naturally came to overlap. While the EPR effect is not universal, especially given the heterogeneity of human tumors, it is nonetheless a powerful tool for passively targeting certain leaky tumors, and its benefit to improving the immune landscape of these tumors should not be discounted.1–3 Just as clinicians could not give systemic small molecule chemotherapeutics to patients and elicit a favorable anti-tumor response, researchers could not effectively eliminate tumors with nanoformulations. However, the combination


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of nanoparticle-mediated remodeling of the tumor microenvironment has allowed cancer immunotherapy to make breakthroughs in how we understand and treat cancer. In this review, we lay out the physical and chemical components of the tumor microenvironment, the challenges to therapy they present, and how nanotechnology has been used to circumvent these challenges and enhance cancer immunotherapy. We hope to illustrate to readers that targeted therapy gives clinicians flexibility to control the tumor microenvironment without inducing systemic effects that could result in toxicities. The Tumor Microenvironment and Barriers to Therapy Tumor-Associated Fibroblasts and Collagen One key feature of tumors that presents a huge barrier to therapy is the presence of tumor-associated fibroblasts (TAFs), also referred to as cancer-associated fibroblasts. TAFs arise through the differentiation of mesenchymal stem cells (MSC) during tumor initiation.4 These fibroblasts provide cancer cells with physical support and signaling feedback that is essential to their growth. Fibroblasts produce collagen, fibrin, and other components of extracellular matrix. Fibroblasts and their secretions form thick stroma that supports and protects tumors, isolating tumor nests from the immune system and providing the structure of the tumor microenvironment. Time and time again, fibroblasts have been implicated as a necessary feature of the tumor microenvironment that supports tumor growth. TAFs are responsible pro-inflammatory signaling in the TME


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that results in the recruitment of tumor-associated macrophages,5 as well as vascularization and enhanced tumor growth.6 The presence of fibroblasts results in chemo-resistance in some tumors, both through physically preventing drugs to reach the tumor7 and through signaling pathways that change tumor cell physiology.8 Impeding the cross talk between tumor cells and fibroblasts can have profound effects on tumor growth. Scientists have recognized for decades that the presence of stroma can prevent the activation of a robust anti-cancer immune response.9 For example, inhibiting the IL-4 mediated feedback between cancer cells and fibroblasts can result in tumor rejection.10 Recently, groundbreaking work found that breast cancer subtypes can be influenced by platelet-derived growth factor-CC.11 The secretion of this growth factor within the tumor microenvironment by TAFs resulted in cancer cells displaying the far more aggressive basal phenotype, rather than the less clinically challenging luminal subtype.11 This is a prime example of the devastating effects the tumor microenvironment can have on the aggressiveness of the disease in question and impact patient survival. Myeloid Cells Myeloid cells, primarily macrophages, dendritic cells (DCs), and myeloid-derived suppressor cells (MDSCs), are key players in tumor immunomodulation. Generally, macrophages and MDSCs are immunosuppressive, while DCs vary depending phenotype. NF-B signaling in tumor-associated myeloid cells has been found to


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promote metastasis in lung cancer models.12 MDSCs, which are attracted to the TME through CCL2, thereby preventing an anti-tumor immune response.13 MDSCs have also been known to suppress natural killer cells, another cell type capable of killing tumor cells.14 To this end, specifically targeting MDSCs is currently thought to be a way to boost anti-cancer immunity and responses to immunotherapies. A major subtype of myeloid cells involved in tumor inflammation and vascularization are tumor associated macrophages.5 Macrophages are one of the most abundant cells in the TME, but the role they play is complex. Macrophages can be polarized into M1 or M2 phenotypes depending on their role; roughly speaking, M1 macrophages, the classical phenotype, are pro-inflammatory and generally fight bacterial infections.15 M2 macrophages are the “alternative” phenotype, generally associated with wound healing and immune suppression.16 In healthy systems, they act as a balance to keep immune responses in check. However, macrophage function can be warped within the tumor microenvironment, with macrophages contributing to disease progression. M1 macrophages, as pro-inflammatory agents, are generally seen good for generating an anti-tumor response, because they can recruit lymphocytes. Unfortunately, they are usually less abundant than M2 macrophages, which play an immunosuppressive and pro-tumor role. The differentiation of TAMs from naïve to the M2 phenotype is thought to be controlled by colony-stimulating factor 1.5 Higher blood concentrations of CSF1 correlate with poor prognosis in many cancers, particularly in the


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reproductive system.16 CSF1 is produced by tumor cells in response to feedback loops within the tumor microenvironment. A recent paper suggested that combining CSF1 blockade with checkpoint inhibitors might boost responses to checkpoint inhibitor therapy, as the presence of M2 TAMs are associated with lower concentrations of CTLs within the tumors.5 Generally, TAMs are associated with poor prognosis, due to the relative abundance of M2 macrophages compared to M1 macrophages, although the opposite correlation occasionally found. From the time of cancer onset, TAMs are associated with cancer cells and producing cytokines like TNF, IL-6, and IL-1 to support tumor growth.17 TAMs can also stimulate cancer cells to activate STAT3, which promotes tumor-initiation and immunosuppression through MDSCs. Directly suppressing TAMs early in tumor initiation reduces the ability of tumors to form.18 As cancer cells proliferate, TAMs expand their role in the TME, secreting different cytokines, including VEGF to stimulate angiogenesis in hypoxic areas and MMPs to assist in invasion and metastasis.17,19 TAMs also begin secreting cytokines like IL-10 to suppress CD8+ T cell responses and stimulate the recruitment of Tregs. Macrophages are even implicated in chemoresistance, assisting in the survival and regrowth of tumors after treatment.18 Some of this effect comes from TAM-mediated activation of cancer stem cells (CSC), which are key to tumor initiation, drug resistance, and relapse following treatment.20 Dendritic cells are a class of antigen-presenting cells that may play either an immune-activating or immune-suppressing role in the tumor microenvironment in


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response to a number of factors. They may also be derived from a myeloid (mDC) or a plasmatoid (pDC) background, and mature in response to toll-like receptor activation, although they vary slightly by function. mDCs are IL-12 secreting cells and mature in response to MHC II and CD80/CD86 costimulation, while pDCs respond to viral threats through the secretion of type 1 interferon. In the context of cancer, stimulatory DCs support anti-tumor effector cells, such as T cells and NK cells, within the TME. Type 1 interferon stimulates antitumor activity of CD8+ dendritic cells, which then prime CD8+ T cells to recognize tumor tissue.21,22 Many proposed cancer vaccines and other immunotherapies depend on the ability of stimulatory DCs to uptake, process, and present antigens to educate T cells. DCs are even used in ex vivo expansion of tumorspecific T cells or are themselves educated ex vivo and injected into patients.23 However, in immunosuppressive conditions, such as those in the tumor microenvironment, the ability to mature and process and present antigens is inhibited in some DCs,24 resulting in a tolerogenic cell type. Tolerogenic DCs are also known to produce IL-10, rather than IL-12, and thereby activate regulatory T cells.25 Mature tolerogenic DCs can both stimulate Tregs and suppress other T cell population expansion, including cytotoxic T lymphocytes (CTLs).25,26 Tumor-Infiltrating Lymphocytes The ability of lymphocytes to penetrate a tumor is a key factor in evaluating patient prognosis. Typically, CD8+ T cells, CD20+ B cells, and NK cells are associated


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with positive patient prognosis.27–30 In many cancers, including breast cancer, melanoma, ovarian cancer, and colorectal cancer, more tumor-infiltrating lymphocytes consistently correlate to better patient outcomes. They are currently used as a biomarker and a method of evaluating the likelihood of patients to respond to certain therapies.31,32 The presence of TILs, evaluated through biopsy histology, is used to classify tumors as “immune-permissive” or “immunosuppressive” and has a great deal of bearing on whether patients respond well to therapy, particularly immunotherapy, like checkpoint inhibitors. Melanoma-based studies found that patients with “moderate and marked diffuse infiltrate” of lymphocytes had over 93% five-year survival, compared to approximately 78% five year survival in patients with no TILs.33 In breast cancer, TILs are a key feature across subtypes in determining patient prognosis and response to chemotherapy.34,35 The more TILs in a triple-negative tumor at the time of diagnosis, the better the patient outcome is expected to be, based on extensive studies of patient clinical data.32 In HER2+ breast cancers, higher concentrations of TILs at the time of diagnosis was predictive of a better response to both anthracycline and trastuzumab.36 In addition, TILs are associated with lower rates of cancer recurrence.37 Much of this data indicates that there is an immune component to many cancer therapies, even those not classified as immunotherapies. More immune permissive tumors are more susceptible to immunotherapy because activated T cells and natural killer (NK) cells have better access to tumor cells; immunosuppressive tumors restrict lymphocytes to the outer edges of


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the tumor and/or kill lymphocytes that do manage to penetrate the tissue barriers. Because of this, immunotherapies, which act by stimulating immune cells to recognize and kill tumor tissue, are ineffective in immunosuppressive environments, because lymphocytes cannot access the tumor. Increasing the number of TILs is a key piece of TME modulation that is currently being studied to improve patient outcomes and expand the number of patients that may benefit from immunotherapies. Of course, as might be expected, the role of lymphocytes in tumors can be complicated. While CTLs and NK cells are desirable for their anti-tumor activity, some lymphocytes that accumulate in tumors are detrimental to therapy. Regulatory T cells (Tregs) are often immunosuppressive and work to promote tumor growth and inactivate CTLs. They are often recruited to tumors after the infiltration of CTLs via CCR4-binding cytokines such as CCL22.38 IL-2 secreted within the tumor microenvironment then supports the function of Tregs as they are recruited and enhances their production.38 Tregs are also IL-23 receptor positive; the binding of IL-23 to IL-23R on Tregs stimulates Stat3 activation in Tregs, which triggers the production of IL-10, an immunosuppressive cytokine.39 Tregs may act in both antigen-specific and antigen-independent manners.40 Antigen-independent Tregs are capable of broadly suppressing CTLs; antigen-specific Tregs suppress only subtypes of CTLs that are specific for the same antigens.41 Antigenspecific Tregs are the most potent suppressors of CTLs, as they are responsible for maintaining self-tolerance. These distinctions are important to immunotherapy for a


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number of reasons; one, because systemic reduction of antigen-independent Tregs can result in autoimmune disease, and two, because in developing cancer vaccines it may be possible to choose antigents that result in weaker antigen-specific Treg responses, as the strength of Treg activities has been noted to vary by antigen.42 Because of the strength of Treg immunosuppression, the ratio of CTLs to Tregs is often necessary to understand the character of the immune microenvironment within tumors and assess patient responses and prognosis.43 In fact, Tregs correlate with lower overall survival across cancer types.44 Therefore, reducing Tregs within the tumor is a promising avenue of research for immunotherapy. Nanoparticle-Based Strategies to Enhance Immunotherapy Immunotherapy is defined a treatment that acts on the immune system rather than primarily acting to kill cancer cells, as compared to chemotherapies, which kill rapidly dividing cells. The benefits of immunotherapy are myriad, especially compared to traditional therapies, but they have significant shortcomings. The biggest drawback, currently, is the fact that only a small fraction of patients respond to immunotherapy, somewhere around 10%. Additionally, immunotherapies are only approved for just under a third of cancers. Response varies a great deal by the cancer type—recent studies have found that drugs like ipilimumab and nivolumab, a CTLA-4 and a PD-1 checkpoint inhibitor, respectively, given in combination to patients with late-stage melanoma, can extend progression-free survival from as low as 3 months on


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monotherapy to a year or more.45 The median overall survival was over 18 months, and was not reached by the end of the study. These results were seen in patients that were both PD-1 positive and PD-1 negative. Tumors also shrank to approximately half their original size, on average, on combination therapy. However, even with these remarkable results, which required an average of four doses, 38% of patients on the combination therapy who left the study did so because of toxic side effects. Severe or life-threatening adverse effects (grade 3 or 4) were reported by 55% of patients in the combination therapy group, and 88% of combination therapy patients experienced adverse events of some kind. By comparison, only 58% of patients responded to combination therapy.45 Most immunological adverse reactions, such as diarrhea, colitis, and flu-like symptoms, could be resolved, but endocrine-related effects could not. Most strikingly, all these challenges were found in melanoma, which has one of the best response rates to immunotherapy. Obviously, the extension of patient’s lives is a huge benefit of immunotherapy, and it has offered select patients a much better chance of achieving remission than current therapies.46 However, the realities of immunotherapy treatment regimens leave a lot to be desired with respect to patient experience and response rate.47 Immunotherapeutics make up a huge proportion of current clinical trials, as researchers look for new ways to activate the immune system against cancer. The challenge going forward will be to develop smart strategies for combining therapies to deliver a safe,


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robust response. Oftentimes, the tumor microenvironment provides a many-layered barrier to immunotherapy that goes far beyond the expression of PD-1 or CTLA-4. One way to address the current needs is to turn to nanomedicine. Nanomedicine offers the ability to target therapies to specific cell types or organs, rather than systemic administration that may result in side effects. Oftentimes, tumors hijack the body’s natural responses to infection or wounds and use them to promote tumor growth, as discussed above. And while developing treatments to reduce the activity of Tregs, for example, might be tempting to reduce the immunosuppression in a tumor, it would also leave the patient at risk of unbalanced immune reactions to every infection or wound, large or small, they may incur while on the treatment. It even leaves patients at risk of autoimmune disease. Many current experimental uses of nanoscience try to ameliorate these risks by targeting therapies to the tumor microenvironment by using targeting ligands or specially developed biomaterials, thereby limiting systemic exposure. Nanoparticles often rely on targeting to selectively engage certain cell types. While this is also true of antibodies, nanoparticles have several advantages over antibodies in many contexts. First, because nanoparticles are so varied, they have the ability of delivering far more types of therapeutics than antibodies, because they can be used to encapsulate insoluble materials or cargo at high risk of degradation, like nucleic acids. The payload per particle is also often higher for nanoparticles than for antibody-drug conjugates,


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which require specialized chemistry to conjugate cargo in such a way that function is not inhibited.48 Antibodies are often conjugated to nanoparticles for targeting, giving researchers the benefits of each—high target selectivity and high therapeutic loading and retention in tunable structures. Many nanoparticles are also targeted using much smaller moieties than antibodies, such as small molecules, peptides, or even portions of antibodies. This often allows for selectivity at a fraction of the cost of antibodies, a powerful benefit in cancer research, where the cost of therapy is already sky-high. Using targeted therapy may also allow for higher tolerated doses, because they interact less with the rest of the body. Indeed, one of the most successful cases of nanoformulations in the clinic is the liposomal form of amphotericin B; systemic amphotericin B is linked with severe nephrotoxicity and other side effects that limit its use in patients. The nanoformulation of amphotericin B into liposomes or similar constructs improves the tolerance and allows for higher doses.49 Nanoscience can help improve immunotherapy in a similar fashion; by treating local immune microenvironments, rather than systemically, we can make therapy safer. In addition, nanomaterials often allow for deeper tumor penetration of cargo than biologics, which are heavy components of immunotherapy.1–3 New advances in nanoparticle engineering allow for deeper tumor penetration than the EPR effect allows.50 With enhanced tumor penetration, full remodeling of the TME is possible, giving nanoscience an advantage over therapeutics, like antibodies, that may only reach the edges of tumors, which is


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often highly desmoplastic. There is a large body of work available showing the potency of utilizing nanoscience to reshape the tumor microenvironment, which may result in better responses to immunotherapy; the nanoformulations used in cancer immunotherapy are listed in Table 1 and explored in the text below. In addition, several recent reviews have outlined the benefits of using nanoparticles for cancer immunotherapy from a materials standpoint, as many nanomaterials are particularly well suited to enhance immune responses.51–53 Table 1. Nanoformulations for cancer immunotherapy. ROS=reactive oxygen species; RNS=reactive nitrogen species; ICD=immunogenic cell death; TAFs=tumor associated fibroblasts; TAMs=tumor associated macrophages; TME=tumor microenvironment; APCs=antigen-presenting cells; DCs=dendritic cells; NK cells=natural killer cells; STING=stimulator of interferon genes.

Nanoparticle Metal

Metal Oxides Lipid

Liposomes

Mechanism ROS- and RNSproduction, protein binding, cytokine disruption, nanovaccine, photodynamic therapy, ICD ROS production, M1 activation RNAi, Trap expression, sTRAIL expression, chemotherapy, nanovaccine, immune activation Chemotherapymediated TAM depletion, VEGF knockdown, TAFdepletion, STING pathway activation,

TME Target Vessels, TAFs, TAMs, TME, APCs, tumor cells

Citation 84, 93-96, 108, 121, 135, 138, 157

TAMs

84, 85

TGF--expressing tumor cells, CXCL12, Wnt-5a, IL-10, CXCL13, fibroblasts and tumor cells, APCs TAMs, TAFs, NK cells, DCs, tumor cells, APCs, tumor cells

56, 58, 59, 63, 64, 96, 104, 115, 124


72, 80, 81, 102, 126, 118, 153

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Polymer

Nucleic Acid

Peptide/protein

Cell-based Carbon nanotubes Silica Zinc phosphate

ICD induction, nanovaccine, ICD Nucleic acid delivery, TAM depletion, chemotherapy, nanovaccine, antigen presentation, ICD RNAi and TAM depletion, TNF downregulation, nanovaccine, ICD Chemotherapy, photoimmunotherapy, nanovaccine, ICD Checkpoint inhibition, chemotherapy, ICD Nanovaccine Nanovaccine, ICD ICD

VEGF-expressing tumor cells, TAMs, TAFs, APCs, T cells, tumor cells

57, 71, 97, 101, 102, 105, 109, 113116, 118, 121, 132, 155, 156

TAMs, APCs, tumor 80, 87, 106, 125, cells 131, 161

TAFs, APC, tumor cells

100-102, 119, 149, 155, 159

Tumor stem cells, tumor cells APCs APCs, tumor cells Tumor cells

69, 82, 154 108, 133, 134, 136 26, 152 158

Modulating Cytokine Levels Cytokines are the mechanisms through which cells within the tumor microenvironment communicate with each other, and therefore the cytokine profile of a tumor can make or break therapy. Reducing signals that stimulate Tregs or TAMs can reduce immunosuppression, increasing cytokines that recruit CTLs and improve T-cell infiltration. Nanoscience offers us a number of ways to shift cytokine profiles. One effective way of modulating the cytokine profile of the tumor microenvironment is to reduce cytokines. Most often, this occurs through gene therapy of some kind, either gene knockdown or gene transfection. For example, Xu et al. found in 2014 that using nanoparticles to deliver siRNA against transforming growth factor-


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(TGF-, reduced immunosuppression within the tumor microenvironment sufficiently to improve the efficacy of a melanoma vaccine in a B16F10 model.54 They identified TGF- as the primary cytokine that was elevated in late-stage tumors that did not respond as well to the vaccine compared to early stage tumors.54 After TGF- knockdown, the number of tumor-infiltrating lymphocytes increased in vaccinated mice and tumor burden remained low.54 RNAi has also been used in tumors against pro-angiogenic vascular epithelial growth factor (VEGF) to reduce tumor vascularization.50 Knocking down VEGF within tumors had the effect of significantly reducing tumor volume for at least nine days after administration by limiting angiogenic signaling.55 It has been noted for decades that VEGF inhibition greatly improves immunotherapy, as anti-angiogenesis and normalized vascularization increases CTL recruitment. Nanoscience has been key to RNAi, and especially to VEGF knockdown, because it provides and effective method of both protecting siRNA from RNases and to release siRNA into cellular cytoplasm. Recently, more innovative approaches to cytokine regulation within the TME have also been developed. Protein trap therapy has been applied to multiple cancer models and cytokine profiles with wide success, and relies on nanoparticles to selectively deliver the genetic material.56 Plasmid DNA encoding for a small, antibody-like trap proteins are encapsulated into lipid nanoparticles with either a calcium phosphate (LCP) or DNA/protamine (LPD) core. These nanoparticles are decorated with a PEG layer for stealth and targeting moieties to target desired cell types are conjugated a subset of the


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PEG polymers. So far, these particles have been shown to target tumor cells through a sigma receptor and hepatocytes through a galactose receptor.56,57 The mechanism for trap delivery and expression is shown in Figure 1. In addition, multiple traps have been delivered to the TME, depending on the cytokine profile of the tumor.53 The delivery of a trap against CXCL12 was used to prevent liver metastasis in colorectal cancer models after CXCL12 was identified as a key cytokine in priming liver metastasis.57 Plasmid DNA encoding for CXCL12 trap was delivered to hepatocytes using galactose-decorated LCPs. Hepatocytes expressed this plasmid for 3-5 days, on average, with the strongest expression on day 2.57 However, the effects of CXCL12 treatment were robust and longlasting, protecting the liver from metastasis for over three weeks after the final treatment. It also extended the overall survival of mice in a breast cancer liver metastasis model to nearly twice that of the untreated control group.57 In addition to blocking metastatic signals, blocking and trapping CXCL12 in the liver also suppresses the recruitment of other CXCR4+ immunosuppressive cells, such as Tregs and MDSCs.57


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Figure 1. Trap therapy for cytokine modulation. (1) Lipid-based nanoparticles containing condensed plasmid DNA and decorated with functionalized PEG binds to target cell surface receptors. (2) The cargo is released from the endosome into the cytoplasm. (3) Plasmid DNA is transported to the nucleus. (4) The DNA is transcribed and translated into traps, which are then secreted. (5) Traps bind with cytokines and remodel the TME.

Since the success in blocking metastatic signaling, traps have also been used in reducing immunosuppression in established tumors. In Braf-mutated melanoma, Wnt family member 5A has been associated with DC tolerization, metastasis, fibrosis, and CTL suppression.58–62 Melanomas treated with Wnt5A trap were significantly less fibrotic and more apoptotic than untreated tumors.62 Wnt5A trap was also administered in combination with low dose doxorubicin, which acts as an inducer of immunogenic cell death, which is discussed in more detail below.62 Activating the immune system against the tumor through ICD and enhancing CTL infiltration by trapping Wnt5A resulted in


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significant reduction of tumor growth for weeks after treatment and significantly prolonged survival.62 Checkpoint Inhibitors and T Cell Stimulators Checkpoint inhibitors have been a hot trend in cancer research since the approval of ipilimumab and the widely-publicized use of pembrolizumab on President Jimmy Carter. However, as discussed in the introduction to this section, checkpoint inhibitors are not effective in most patients, and may cause significant side effects when they are. For this reason, researchers in nanoscience are developing novel ways to decrease checkpoint inhibition in tumor models that may prove to be more widely effective and safer. Traditional checkpoint inhibitors work by physically blocking PD-1, PD-L1, or CTLA-4 on the surface of target cells. Some nanoparticle-based attempts at checkpoint inhibition preserve this approach, but seek to improve upon it. For example, as the local expression of protein traps can bind cytokines and change the immune profile, so too can they be used to bind and block checkpoint molecules. PD-L1 traps have been applied to primary colorectal cancer treatment in combination with oxaliplatin induced immunogenic cell death, and liver metastasis models of colorectal cancer in combination with a colorectal cancer vaccine and the CXCL12 trap discussed in the previous section.63,64 Trap therapy has also been shown to be more effective and safer than antibody therapy.64 The PD-L1 trap expressed in the liver was more effective than


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the whole protein administered systemically.56 In addition, the PD-L1 trap increased TILs and prolonged CD8+ cell survival in the TME.63 The addition of PD-L1 trap enhanced the efficacy of both the vaccine and CXCL12 trap therapy.63 Similar approaches were taken in CTLA-4 targeting therapies; in studies of a Trp2 melanoma vaccine, transfecting DCs with mRNA encoding for anti-CTLA-4, as well as GITR, reduced the Treg population and boosted the efficacy of the vaccine.65 It was thought that local administration of CTLA-4 blockade made the therapy safer while providing similar immunomodulatory effects to systemic antibody administration.65 Gene therapy has also been used to knock checkpoint molecules down in target cells. Folate-modified PEI nanocomplexes carrying siRNA against PD-L1 have been selectively targeted to folate-receptor-positive ovarian cancer cells. These nanocomplexes were successful in knocking down expression of PD-L1 in vitro.66 Still others found novel ways to deliver checkpoint inhibitors to the TME as proteins, rather than relying on transfection. One research group developed fusion proteins with the ability to bind both PD-L1 and TGF-, which delivers a dual effect in TME remodeling.64,67 The suppression of TGF- signaling reduced proliferation and chemoresistance while PD-L1 blockade reduced immunosuppression.67,68 Multifunctional nanoparticles that both display PD-1 and deliver IDO inhibitors to the tumor microenvironment have also been developed. Nanovesicles harvested from PD-1 expressing cell lines carry PD-1 on their surface, and can be loaded with 1-


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methyltryptophan, an inhibitor of DC immunosuppressive molecule IDO.69 This dualapproach to reducing immunosuppression led to significant improvement in CTL infiltration and tumor growth inhibition.69 Alternatively, anti-CTLA-4 antibody has been encased into a mesoporous silica nanoscaffold that can be injected locally.70 This antibody-loaded scaffold slowly releases anti-CTLA-4 directly into the TME, which produces a stronger and more prolonged tumor growth inhibition than the traditional systemic injection of anti-CTLA-4.70 Still other techniques outside of checkpoint inhibitors exist as a way of controlling T cell activity within the TME. For example, microparticles encapsulating mTOR inhibitor rapamycin can create a tumor-targeted depot, slowly releasing rapamycin throughout a vaccine regimen. Low doses of rapamycin supported the formation of central memory T cells and increased the proliferation of antigen specific T cells for extended periods of time.71 Tumor-Associated Macrophage Inhibition and Remodeling Tumor-associated macrophages are major immunosuppressive players in the tumor microenvironment, as detailed above. M1 phenotypes, which bolster ant-tumor responses, are often suppressed in the TME, though their prescence can be a hallmark of promising prognosis. Tumors often stimulate macrophages to take on an M2 phenotype, which supports tumor growth through production of cytokines like IL-10. For this reason, many avenues of TME modulation are centered on the inhibition of


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TAMs as a way to increase the efficacy of cancer treatments. TAM depletion is popular; many approaches utilize targeted approaches to deliver drugs, like doxorubicin,72 toxins,73,74 or phototherapeutic agents75 to TAMs and specifically reduce their numbers within the tumor. TAMs can be targeted using mannose, which binds to mannose receptor on TAMs and DCs, specific peptides such as M2pep, alendronateglucomannan, or CD206 antibodies with good success.72–75 As TAMs have been associated with drug resistance, angiogenesis, and immunosuppression, and TAM depletion has been shown to improve response rates and tumor regression due to the lack of TAM-associated immunosuppression.72–75 TAM modulation is particularly important because the pro-tumor activities of TAMs, including angiogenesis and the recruitment of Tregs, is not limited to the initiation of tumors. TAMs have also been implicated in the recurrence of tumors postantiangiogenic therapy.19,76,77 The same trend was found in the administration of iRGD, a cyclic peptide developed to enhance tumor penetration. While iRGD was effective at increasing drug uptake and reducing tumor volume, relapse still occurred and was attributed to TAM-mediated revascularization.78 Going forward, the peptide was modified to nRGD, which contained a short, macrophage-specific sequence, AAN. This bifunctional peptide could not only enhance tumor penetration, but could also specifically inhibit TAMs.73 This reduction in TAMs, illustrated in Figure 2, correlated with enhanced anti-tumor activity of drugs co-administered with nRGD, such as


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doxorubicin.73 The increase in efficacy could be attributed to an overall shift in cytokines the TME, such as a reduction of IL-10 and TGF-, and normalization of the tumor vasculature, both of which could be attributed to the reduction in TAMs.73 TAM depletion has also been shown to suppress metastasis in triple negative breast cancer and melanoma models.79

Figure 2. TAM depletion via nRGD/doxorubicin liposomes. Nanoparticles targeted with nRGD accumulate in tumors and bind to TAMs, vessels, and tumor cells as nRGD is slowly degraded (degradation phases shown sequentially in structures 1-3). This TAM and tumor cell targeting leads to remodeling of the TME and tumor growth inhibition as IL-10 production is reduced and TNF and IL-6 is increased.

Still more selective TAM-targeted mechanisms have been developed, such as M2pep.80 M2pep selectively binds M2 Tams over M1 TAMs and other leukocytes, which


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reduces the risk of systemic immune side effects that may arise by depleting the entire patient of all macrophages. In addition, by selectively targeting M2 macrophages, M1 macrophages may remain present in the tumor, which is important for tumor growth inhibition. M2pep has been conjugated to proapoptotic peptides like KLA as a way of selectively killing M2 TAMs while leaving the anti-tumor M1 macrophages and other immune cells within the TME intact. The selective reduction of M2 TAMs improved overall survival of tumor-bearing mice.80 M2pep has also been applied to siRNA delivery systems, in which M2pep was used to deliver VEGF RNAi to macrophages in lung cancer models.81 M2pep-mediated VEGF knockdown reduces both VEGF and TAMs in the TME and improves overall survival.81 All this underlines the key role TAMs play in the TME, and how the reduction of immunosuppressive TAMs, regardless of how specific, can result in favorable remodeling of the TME and, by extension, tumor inhibition. However, many researchers also see TAMs as a powerful resource in fighting cancer. In seek to rid tumors of macrophages, some researchers are using the macrophages within the TME to their advantage. Low doses of radiation or chemotherapeutics have also been shown to shift TAMs from M2 to M1 phenotypes.82 The M1 phenotype has been shown to not only reduce immunosuppression and inhibit tumor growth, but also to reduce fibrosis in a tumor, further reducing the tumor’s defenses.82 Alternatively, metal nanoparticles, like iron oxide, gold or silver, have been used to shift the TAM phenotypes from M2 to M1 by inducing reactive oxygen species


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in situ.83–85 Metal nanoparticles have been associated with intrinsic anti-tumor activity and an increase in oxidative stress, which stimulates the activation of M1 macrophages over M2, causing phenotype switching and increasing the number of M1 macrophages in the tumor. Shifting M2 macrophages to M1 has been associated with tumor growth inhibition and significant TME remodeling, including shifts in the concentrations of TNF, IL-10, and TGF-.83–85 Some groups have also been using intracellular modulation to shift TAM phenotypes; in Huang et al. (2016), let-7b, a synthetic miRNA mimic, was targeted to TAMs and DCs through the mannose receptor and resulted in the reduction of IL-10 expression, an increase in anti-tumor TAM and DC activation, and overall tumor inhibition.82,86 The endocytic properties of macrophages have also been taken advantage of to prompt the uptake of dexamethasone DNA nanotubes, which then stimulated TAMs to significantly remodel the TME. TAMs treated with nanotubes downregulated production of TNF and expression of ICAM-1 and VCAM-1 on tumor vessels, which reduced the recruitment of immunosuppressive cell types.87 Targeting Tumor-Associated Fibroblasts Tumor-associated fibroblasts are induced from stroma by cancer cells to support their survival, proliferation, and metastasis.88–90 They also form barriers to all forms of treatment, from small molecules to nanoparticles.91 However, research increasingly shows that nanoparticles are the best tool for circumventing tumor fibrosis. Some nanoparticles, like gold nanoparticles (AuNPs), have the ability to suppress fibrosis


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through interactions with proteins in the TME. For example, AuNPs bind VEGF, fibroblast growth factors, and heparin binding growth factors, preventing both angiogenesis and fibrosis.92,93 AuNPs bind a number of key players in both paracrine and autocrine signaling in the TME, including IL-8 and TGF-, and induce ER stress that leads to changes in the secretome of both cancer and stellate cells.94,95 The mere presence of AuNPs in pancreatic tumors is enough to reduce and shift the interactions between fibroblasts and cancer cells, resulting in tumor growth inhibition and a remodeled TME that is more susceptible to treatment.95 The physical barrier formed by fibroblasts against cancer therapy is often twofold; the dense, fibrin-rich, desmoplastic extracellular matrix produced by the fibroblasts makes it difficult for therapeutics to penetrate the tumor, and the fibroblasts themselves take up many of the therapeutics themselves, shielding tumor cells from their effects. Some researchers have taken advantage of fibroblasts’ high rate of uptake to target TAFs and remodel the TME. Numerous TAF-specific gene therapy nanovehicles have been developed.58,96,97 For example, Miao et al. used lipid nanoparticles to transfect fibroblasts with plasmid DNA encoding for a secretory form of TNF-related apoptosisinducing ligand (sTRAIL). TRAIL, as its name indicates, stimulates apoptosis in tumor cells, and is therefore a promising therapeutic agent, especially when produced by local TAFs into the TME, as shown in Figure 3.58 By transforming TAFs into the machinery that produced therapeutics, a two-fold effect occurred in bladder cancer and pancreatic


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cancer models; tumors shrank as tumor cells underwent apoptosis, and proliferation and resistance was inhibited as fibroblasts underwent quiescence, ceasing in their support of the immunosuppressive and pro-cancer TME.58 Similarly, the same group was able to use lipid-protamine-hyaluronic acid nanoparticles (LPH) to deliver siRNA against Wnt16, a driver of -catenin and a key signaling molecule in TME immunosuppression, to fibroblasts, interrupting the supportive paracrine signaling pathway between fibroblasts and tumor cells.96 This knockdown of Wnt16 so drastically impeded fibroblast-tumor cell interactions that the tumor became more susceptible to platinum therapy, administered in a lipid-based nanoformulation, significantly impacting treatment outcomes.96 By supplementing cisplatin Figure 3. LPDs transfect fibroblasts, harnessing desmoplastic conditions to produce soluble TRAIL and kill neighboring tumor cells.

administration with Wnt16 knockdown, cisplatin was

able to penetrate the tumors more deeply and the tumors were unable to develop resistance to platinum over time, as compared to tumors treated with cisplatin alone.92


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The same trend may be possible for CTLs, making TAF remodeling a potent partner for immunotherapies. Tumor-associated fibroblasts can be harnessed for therapy in other ways as well; for example, rather than transfecting TAFs, Ji et al. developed a peptide-based nanocarrier that could be degraded by fibroblast activation protein-, which is highly expressed on TAFs.98 The inducible instability of this nanoformulation allowed for the targeted release of hydrophobic anti-cancer drugs into the TME, taking advantage of the high concentrations of TAFs to achieve highly specific drug delivery.98 Still, many researchers see fibroblasts as such a significant hindrance to therapy that they seek to simply rid tumors of fibroblasts, rather than try to co-opt them for therapy. A number of TAF-depleting nanotherapies exist, including photodynamic therapy targeted against FAP,99 drug-loaded hydrogels,100 and TAF-targeted chemotherapy.101,102 TAF-targeted therapies most often utilize TAF-specific proteins, like FAP, or overexpressed receptors, like fibroblast-growth factor receptor, with good selectivity, an important feature in nanomedicine.97,99 Notably, the method of depletion may dictate the impact TAF depletion had on the tumor; for example, losartan-loaded hydrogels were able to significantly reduce the amount of collagen and the number of TAFs within an orthotopic model of triple-negative breast cancer, but this TAF-deficiency was not sufficient to suppress tumor growth. The lack of TAFs did, however, make the tumors more susceptible to chemotherapeutics, and the combination of losartan-loaded


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hydrogels and liposomal doxorubicin was able to significantly reduce tumor growth and prolong survival.100 In contrast, photoimmunotherapy directed against FAP+ TAFs was shown to selectively kill fibroblasts and not tumor cells, but did result in long-term inhibition of tumor cell proliferation.99 The tumor growth inhibition was associated with greater CTL infiltration in TAF-depleted tumors.99 While both studies used a 4T1 triplenegative breast cancer model, among others, one caveat against a direct comparison of the two is that the hydrogel study was undertaken in an orthotopic model, and the PDT study was undertaken in a subcutaneous model, which is notoriously insufficient to predict translatable results. Regardless of whether TAF depletion alone is capable of inhibiting tumor growth, evidence is clear that TAFs are a major barrier to effective cancer therapy and TAF modulation can dramatically improve outcomes in animal models. Immune Stimulation Nanomedicine has been effectively used to target specific TME cell types to deliver therapeutics, change their function, or specifically inhibit them, but it has also been successfully used to raise anti-tumor immune responses. Cancer Nanovaccines One of the most common and successful ways of stimulating an anticancer immune response is the cancer nanovaccine. Entire reviews have been written on nanovaccines, and even on subtypes of nanovaccines, so we cannot cover the full


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breadth of the topic here, but we will try to provide a sufficient understanding of how cancer nanovaccines fit into the landscape of TME modulation. Cancer nanovaccines are nanovehicles for cancer-associated antigens and adjuvants that increase the stimulation of antigen presenting cells, such as DCs, and raise an immune response against tumors. They are incredibly versatile and nearly all types of nanoparticles have been used as nanovaccines, including: chitosan,103 lipids,104 polymers,105 nucleic acids,106 metal,107 and carbon.108 Nanovaccines are more attractive than traditional vaccines because they can deliver a smaller amount of antigen and adjuvant but still elicit a robust immune response due to their ability to target to lymph nodes and/or APCs. Additionally, because nanoparticles provide protection for their cargo, antigens can be given as peptides, RNA, or DNA.103,104,109 This flexibility makes nanovaccines ideal for raising immune responses against whole proteins encoded by nucleic acids rather than single peptides, which may lead to more robust immune responses. Nanovaccines are also often used to deliver novel adjuvants that target specific immune pathways based on the desired immune response, giving them far more specificity. Pattern-recognition receptor and STING-pathway agonists are common adjuvants in nanovaccines, and some nanomaterials have inherent adjuvant properties.110–113 For example, galactosyl dextran-retinal nanogels loaded with a cancer-associated antigen rupture endosomes and produce ROS.114 Endosomal burst stimulates antigen presentation, making the particle an adjuvant for the antigen it contains, and has been taken advantage of in


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many types of polymeric nanoparticle vaccines.114–116 The simplest vaccines may be one antigen and one adjuvant, or even one antigen in a nanoparticle that has intrinsic adjuvant properties. More elaborate platforms may include multiple antigens or even whole tumor lysates in order to elicit a more robust response, as tumors are notoriously heterogeneous.117 Oftentimes nanovaccines are combined with checkpoint inhibitors or other immunotherapy in order to boost CTLs in the tumor and sustain their survival.104,118 Occasionally one nanovehicle is used for both a vaccine and a therapeutic carrier, such as photoimmunotherapy119 or RNAi.113 For a more thorough, but concise, overview, we recommend Zhu et al. (2017).120 Some researchers have sought to elicit a response similar to nanovaccination by bypassing DC activation and instead using nanoparticles as artificial APCs. Antigencapturing nanoparticles display antigens captured from tumor lysates on their surface and can be used to stimulate T cell activation, a more direct approach than delivering antigens to APCs and trying to stimulate their presentation.121,122 The nature of the captured antigens and the T cell response can be controlled through the surface coating of these antigen-capturing nanoparticles, which makes them a promising avenue for stimulating cancer immunity. Targeted Delivery of PAMPs Over the course of human evolution, the innate immune system has developed a number of methods of recognizing threats and addressing them appropriately; one of


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these methods is through the use of molecular pattern recognition receptors, or PRRs. Molecular patterns that act as ligands to these receptors are known as pathogenassociated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), and include tell-tale signs of cellular infection, such as DNA or dsRNA in the cytosol.123 PAMPs may also be known as microbe-associated molecular patterns, or MAMPs. Each various form of PAMP or DAMP stimulates one or more specific immuneactivating pathway, based on the threat detected. In recent years, the ability of certain PRR pathways in the cell to induce an antitumor immune response has gained attention and birthed a rising segment of cancer nanomedicine research. One of the most promising methods of inducing an antitumor immune response is through the delivery of dsDNA analogues like cGAMP and CpG to induce the stimulator of interferon genes complex (STING) or Toll-like receptors, commonly TLR9. A brief summary of PRRs and immune stimulation is provided in Figure 4.


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Figure 4. PAMPs, PRRs, and Immune Activation.

Many PAMPs are used as adjuvants to improve vaccine activity,110,124,125 but they have also been used effectively alone to stimulate responses against immunogenic tumors. The stimulator of interferon genes (STING) pathway acts by releasing many proinflammatory molecules like interferon that stimulate natural killer cells and dendritic cells, resulting in an anti-tumor immune response. For example, simply loading pHsensitive PEGylated liposomes with c-di-GMP, a STING ligand, and administering them intravenously to B16-F10 metastatic melanoma bearing mice could activate NK cells sufficiently to suppress the formation of metastases.126 Similar tumor suppression was found when liposomal cyclic guanosine monophosphate–adenosine monophosphate


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(cGAMP), another STING agonist, was combined with checkpoint therapy in the B16-F10 metastatic melanoma model.127 Far more common than activating STING, at this point, is using CpG to stimulate TLR9, another nucleotide sensor in immune cells. CpG is a dinucleotide of unmethylated cytidine and guanosine, joined through a phosphate bond, which are often associated with microbial DNA. TL9 activation also stimulates DCs and macrophages to produce proinflammatory cytokines and activate CTLs, and so CpG is often used as a vaccine adjuvant in cancer vaccine research, and has been for some time.26,124,128–130 Initially, CpG was often administered as DNA nanostructures, either as monotherapy or in combination with vaccines and chemotherapeutics.131 Nanoscience has improved the use of CpG over time as an immunostimulatory factor in a number of ways; coadministration of CpG with polyethylenimine (PEI) improves cytokine induction, compared to CpG alone, for instance, particularly with respect to IFN-.132 This improvement of activity was applied to a tumor model in a graphene-oxide-PEG-PEI nanoparticle, which, when injected intratumorally, could suppress tumor growth. The effects were amplified when combined with photothermal therapy, mediated by the graphene oxide.133 Similarly, CpG has also been loaded into carbon nanotubes, copper sulfide nanoparticles, and gold nanoparticles for enhanced inflammatory responses, which also inhibited tumor proliferation when combined with photothermal therapy and/or chemotherapeutic agents.134–137 CpG-loaded AuNPs combined with doxorubicin


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and irradiation even showed some signs of tumor shrinkage, a profound antitumor response.135 Even passive targeting of macrophages with CpG-conjugated AuNPs significantly increases immune stimulation over free CpG and provides survival benefit and controls tumor proliferation.138 Recently, CpG delivery platforms have become more elaborate, including co-encapsulation of CpG and anti-PD1 antibodies to both stimulate an anti-tumor immune response and circumvent immunosuppression simultaneously.139 This combination therapy uses our growing knowledge of the immune system to deliver complimentary therapeutics, a technique that nanomedicine as a field is taking on more and more as we become more interdisciplinary. Carefully designed combination therapies induce much stronger anti-cancer responses, which in turn increases the likelihood of clinical translation.139 Because combination therapies are so promising, the use of nanoscience to carefully co-deliver therapeutics is bound to be a key step forward in the success of cancer immunotherapy. Induction of Immunogenic Cell Death The approaches discussed in this review often work on one particular feature of the immune system, either a cytokine or a cell type, that may then go on to have a wider impact on the tumor microenvironment as a whole; however, some applications of nanomedicine to the field of cancer immunotherapy have been attempting to cast a wider net. Using nanoparticles to induce immunogenic cell death (ICD) in a controlled manner is one of those avenues through which researchers are able to effectively


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stimulate a robust immune response, as shown in Figure 5. Cancer vaccines are often only able to deliver one or two epitopes of cancer antigens at once, although there are a growing number of vaccines built around tumor lysates in an effort to get a more robust immune response. As attractive as a personalized cancer vaccine is, it is labor intensive and promises to be expensive. ICD, especially when combined with nanoparticles, offers a simpler way forward.

Figure 5. Immunogenic cell death.

Many studies have noted that low doses of chemotherapeutics can induce cancer cells to undergo apoptosis in a manner that triggers the release of DAMPs and antigens, called immunogenic cell death.140,141 ICD induction is thought to be part of the reason for the success of metronomic chemotherapy, the process of giving patients small doses of chemotherapy over time, which occasionally resulted in much better responses than the low-dose indicated.142 DAMPs, like PAMPs, act as adjuvants to stimulate the immune system. In this way, ICD inducers essentially act as an in situ vaccine by co-stimulating


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the immune system with tumor antigens and adjuvants.140 However, because many tumor cells are dying and releasing many antigens into the TME, this process provides the immune system with many more antigens than current vaccine strategies (tumorlysate vaccines perhaps excluded), and it is personalized; the antigens released perfectly match the patient’s tumor, rather than the predicted antigens for a mass-produced vaccine. Table 2. Known ICD Inducers with Nanoformulations

ICD Inducer

Nanoformulation

Models

Citation

Doxorubicin

Self-assemble polypeptide, nanocrystals, liposomes, MnO Nanoparticle albumin, Mespoporous silica nanoparticles, PLGA-PEG Polymer, AuNPs

Triple negative breast cancer (4T1), glioblastoma, colorectal cancer Pancreatic ductal adenocarcinoma Pancreatic ductal adenocarcinoma (KPC) Melanoma, triplenegative breast cancer, colorectal cancer Colorectal cancer, hepatocellular carcinoma, breast cancer Colorectal cancer, triple-negative breast cancer

26, 133, 135, 151, 153, 161

Paclitaxel Oxaliplatin

Radiotherapy

Photothermal therapy

Photodynamic therapy

Graphene oxide, carbon nanotube, gold nanoparticles, copper sulfide Metal-organic, zinc pyrophosphate, peptide, MnO


135, 157 152, 155

148-150

133-135 , 137

158-161

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ICD was first recognized as a phenomenon in cancer cells treated with subtherapeutic doses of certain chemotherapeutic drugs, like doxorubicin. The most wellknown ICD inducers include anthracyclines like doxorubicin and mitoxantrone, oxaliplatin, bortezomib, and cyclophosphamide.143 In 2014, a study seeking to validate ICD inducers in vitro and in vivo found eight clinically approved drugs acted as ICD inducers in vivo; daunorubicin, docetaxel, doxorubicin, mitoxantrone, oxaliplatin, paclitaxel, digitoxin, and digoxin.144 However, there is a great deal of contention over docetaxel and paclitaxel in ICD; while they exhibit some hallmarks of ICD, some studies find that they induce a separate kind of immune-modulating cell death that is distinct from ICD.145 Either way, it is becoming clear that the immune system can be stimulated through controlled cell death pathways. Only a subset of these have been used as ICD inducers in nanoformulations; most often they are encapsulated and administered at therapeutic, cytotoxic doses. The interest in ICD as a cancer therapy has even resulted in the synthesis of novel drugs specifically for inducing ICD in cancer cells while remaining safe for healthy cells, like R2016.146 In addition, photodynamic therapy, radiotherapy, and some ultraviolet radiation have been shown to induce ICD.147–150 Nanomaterials like gold nanoparticles are especially valuable for ICD induction because they can enhance photodynamic therapy and radiotherapy intrinsically. While some drugs, like R2016 mentioned above, are being developed to selectively induce ICD in cancerous cells, many methods of inducing ICD were


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developed for other purposes and may have side effects, making them ideal candidates for nanoformulation. Nanoscience has been used to induce ICD in tumor cells and stimulate a local immune response. Nanoformulated doxorubicin has been used to induce ICD in glioblastoma when loaded into monocytes, colorectal cancer in liposomes, triple-negative breast cancer in peptidic nanoparticles, and many other formulations and models.151–154 Lipid-based nanoparticles loaded with doxorubicin has also been used in conjunction with Wnt5A traps, as discussed above in “Modulating cytokines.” Nanoscale cisplatin, in formulations including lipid nanoparticles and organic frameworks, is more effective than free cisplatin in remodeling the TME and stimulating anti-tumor responses through ICD.155 Nano-inducers of immunogenic cell death can even make drug-resistant tumors more susceptible to therapy, which is a huge clinical benefit. Pancreatic cancer, one of the deadliest cancers, can be inhibited by both oxaliplatin and paclitaxel nanoparticles.152,156,157 In addition, nanoparticles can be used to deliver metal or organic nanoparticles for the stimulation of photodynamic therapy (PDT), another known ICD inducer. These therapies often act by inducing significant tumor cell death while also inducing an antitumor immune response that can suppress metastases and T cell memory, compared to chemotherapeutic agents, which often only induce a small fraction of cells to die.158–161 As the field of ICD inducers continues to grow, there is reason to believe that nanoscience will play a key role in making those therapeutics clinically available. For


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example, recent research has shown that single-stranded RNA is able to bind PRRs and induce immunogenic cell death and robust anti-tumor responses. Each ssRNA discovered raised a slightly different immune response, giving researchers the ability to tune therapy to the needs of the tumor.146 However, ssRNA would most likely need to be protected through a nanoformulation in order to be delivered safely to the tumor. This is one of the promises of nanomedicine—making administration of therapies more effective and safer for patients. Current Challenges Although the list of applications of nanomaterials for cancer immunotherapy grows longer by the day, some challenges remain. Preclinical studies undertaken in mice often lack the complexity of human tumors, as the majority are still done using cell lines, which are mostly homogenous cell populations. In contrast, human tumors are heterogenous, with many cells in the same tumor carrying different mutations and the TME of each tumor varying widely from patient to patient. Many immunology studies are also undertaken in subcutaneous models of tumors, which do not accurately portray the immune landscape of orthotopic tumors, let alone human disease.162 This has been particularly troublesome when therapeutics move into clinical trials. Many therapeutics that work in mice will not work in humans at all, as we know from the fact that only 1015% of clinical trials achieve approval.163 Mice are not the best models of the human immune system, but they are the most accessible for researchers, and are likely to


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remain the most common preclinical model. Even with all the work done to ensure that work in animal models is done to the highest standard, it does not mean it will translate well to human patients. Some therapeutics might cause the same effects in mice as in humans, but may come with significant side effects in humans that could not be predicted with an animal model. Humanization of mouse models may help alleviate some errors, but cannot be expected to account for all the intricacies of a clinical setting.164 Not only are the models incomplete, but our understanding of the TME also leaves much to be desired. A great deal of research remains to be done on the components of the TME themselves and their intricate interactions; different tumor systems may respond differently to the same immune manipulations, and until we have a more reliable understanding of the immune interplay in tumors, sophisticated treatments will remain difficult to tailor to tumors. It may be necessary for biomarker testing in clinical trials of nano-immunotherapies, as is currently required of checkpoint inhibitors. In addition, many nanoparticles will require work to scale up safely and reproducibly before they can be tested in humans, which require a great deal more material than mouse studies. This can be a time-consuming process that keeps some therapeutics from entering the clinic quickly, as most nanoparticles are made in a batch process that is not always translatable to industrial needs or standards. Many


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researchers are moving toward making their nanoparticles out of materials that are already generally regarded as safe by the FDA, or in processes that can easily be scaled up, but many are not, and that would slow down the translation of much work currently bridging the gap between nanoscience and cancer immunology. Conclusion As shown in this review, the fields of nanomedicine and cancer immunotherapy are deeply intertwined because of the advantages they offer each other. Nanomedicine allows for the specific targeting of distinct tissues, cell types, or molecules, and can be used to induce stronger responses to ICD inducers than free agents. Going forward, there is room for both fields to continue to grow together and provide more attractive and efficient clinical therapies. Acknowledgements We would like to thank and acknowledge the support of Dr. W. Song in the preparation of this manuscript, as well as the Carolina Center for Cancer Nanotechnology Excellence (NIH grant CA198999) for their support in funding our research. Graphics were created using BioRender.


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