Beyond cancer cells: Targeting the tumor microenvironment with gene therapy and armed oncolytic virus
Authors: Peter Kok-Ting Wan, Anderson J. Ryan, Leonard W. Seymour
Journal: Molecular Therapy (2021)

Cancer gene therapies are usually designed either to express wild-type copies of tumor suppressor genes or to exploit tumor-associated phenotypic changes to endow selective cytotoxicity. However, these approaches become less relevant to cancers that contain many independent mutations, and the situation is made more complex by our increased understanding of clonal evolution of tumors, meaning that different metastases and even regions of the same tumor mass have distinct mutational and phenotypic profiles. In contrast, the relatively genetically stable tumor microenvironment (TME) therefore provides an appealing therapeutic target, particularly since it plays an essential role in promoting cancer growth, immune tolerance, and acquired resistance to many therapies. Recently, a variety of different TME-targeted gene therapy and armed oncolytic strategies have been explored, with particular success observed in strategies targeting the cancer stroma, reducing tumor vasculature, and repolarizing the immunosuppressive microenvironment. Herein, we review the progress of these TME-targeting approaches and try to highlight those showing the greatest promise. Cancer gene therapies are usually designed either to express wild-type copies of tumor suppressor genes or to exploit tumor-associated phenotypic changes to endow selective cytotoxicity. However, these approaches become less relevant to cancers that contain many independent mutations, and the situation is made more complex by our increased understanding of clonal evolution of tumors, meaning that different metastases and even regions of the same tumor mass have distinct mutational and phenotypic profiles. In contrast, the relatively genetically stable tumor microenvironment (TME) therefore provides an appealing therapeutic target, particularly since it plays an essential role in promoting cancer growth, immune tolerance, and acquired resistance to many therapies. Recently, a variety of different TME-targeted gene therapy and armed oncolytic strategies have been explored, with particular success observed in strategies targeting the cancer stroma, reducing tumor vasculature, and repolarizing the immunosuppressive microenvironment. Herein, we review the progress of these TME-targeting approaches and try to highlight those showing the greatest promise. Components of the TME play essential roles in cancer development and growth. In most carcinomas, the malignant tumor cells are supported by a heterogeneous population of non-transformed cells in the TME, including fibroblasts, endothelial cells, immune cells, adipocytes, and pericytes.1Balkwill F.R. Capasso M. Hagemann T. The tumor microenvironment at a glance.J. Cell Sci. 2012; 125: 5591-5596Crossref PubMed Scopus (832) Google Scholar Developing tumors can recruit these stromal cells from adjacent non-tumor tissue and also from the bone marrow to support the emerging neoplastic phenotype.2Lamagna C. Bergers G. The bone marrow constitutes a reservoir of pericyte progenitors.J. Leukoc. Biol. 2006; 80: 677-681Crossref PubMed Scopus (117) Google Scholar Bi-directional communication between the tumor and these microenvironmental cells appears to be critical for promoting tumor progression and metastasis, with tumors secreting cytokines and chemokines that alter surface markers on endothelial cells and induce tumor tolerance.3Motz G.T. Santoro S.P. Wang L.-P. Garrabrant T. Lastra R.R. Hagemann I.S. Lal P. Feldman M.D. Benencia F. Coukos G. Tumor endothelium FasL establishes a selective immune barrier promoting tolerance in tumors.Nat. 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Lin H. et al.The first approved gene therapy product for cancer Ad-p53 (Gendicine): 12 years in the clinic.Hum. Gene Ther. 2018; 29: 160-179Crossref PubMed Scopus (95) Google Scholar and later approaches often exploited the tumor phenotype to regulate a selective cytotoxic effect. However, with the availability of single-cell sequencing and recent insights into tumor heterogeneity and clonal evolution showing the multitude of tumor-associated mutations and phenotypic variation between different regions even of the same tumor,7McGranahan N. Swanton C. Clonal heterogeneity and tumor evolution: Past, present, and the future.Cell. 2017; 168: 613-628Abstract Full Text Full Text PDF PubMed Scopus (976) Google Scholar such "one size fits all" therapeutic strategies now appear less feasible. Accordingly, gene therapy approaches have recently been developed that aim to manipulate the relatively genetically stable TME, including modulation of the local immune system. In particular, progress has been made to deplete cancer stroma and to degrade the tumor vasculature,8Kakarla S. Song X.-T. Gottschalk S. Cancer-associated fibroblasts as targets for immunotherapy.Immunotherapy. 2012; 4: 1129-1138Crossref PubMed Scopus (56) Google Scholar,9Toro Bejarano M. Merchan J.R. Targeting tumor vasculature through oncolytic virotherapy: recent advances.Oncolytic Virother. 2015; 4: 169-181PubMed Google Scholar while cytokine-based gene therapies and vaccines have aimed to boost the antitumor response or suppress tumor tolerance.10Marchand J.-B. Semmrich M. Fend L. Rehn M. Silvestre N. Teige I. Foloppe F. Mårtensson L. Quéméneur E. Frendeus B. BT-001, an oncolytic vaccinia virus armed with a Treg-depletion-optimized recombinant human anti-CTLA4 antibody and GM-CSF to target the tumor microenvironment.Cancer Res. 2020; 80: 5602Google Scholar In addition, armed oncolytic viruses (OVs) are increasingly considered as appealing therapeutic agents because they mediate tumor-selective oncolytic cell-killing mechanisms,11Seymour L.W. Fisher K.D. Oncolytic viruses: Finally delivering.Br. J. Cancer. 2016; 114: 357-361Crossref PubMed Scopus (68) Google Scholar lysing tumor cells to expose proinflammatory signals (pathogen-associated and damage-associated molecular patterns [PAMPs and DAMPs]) and tumor antigens, combined with local expression of encoded immunomodulatory transgenes. This dual mechanism affords many innovations in therapeutic approach. In this review, we consider the most promising TME-targeting strategies using gene therapies and armed OVs, illustrated in Figure 1, and suggest which stromal cells and therapeutic modalities might provide the greatest therapeutic value. Choice of appropriate preclinical model systems is challenging for agents targeting the TME, particularly for immunomodulatory armed OVs, as these are often human selective and do not complete their full life cycle in most rodent cells. Some immune-competent animals, such as Syrian hamsters, are semi-permissive for some OVs,12Dhar D. Toth K. Wold W.S. Syrian hamster tumor model to study oncolytic Ad5-based vectors.Methods Mol. Biol. 2012; 797: 53-63Crossref PubMed Scopus (11) Google Scholar and this provides a handful of tumor models, although it is not clear whether the stromal composition of model tumors or the balance of direct oncolysis and transgene expression has clinical relevance.13Guerin M.V. Finisguerra V. Van den Eynde B.J. Bercovici N. Trautmann A. Preclinical murine tumor models: A structural and functional perspective.eLife. 2020; 9: e50740Crossref PubMed Scopus (28) Google Scholar,14Yamaguchi R. Perkins G. Animal models for studying tumor microenvironment (TME) and resistance to lymphocytic infiltration.Cancer Biol. Ther. 2018; 19: 745-754Crossref PubMed Scopus (10) Google Scholar Similarly, xenografted tumors in transgenic mice provide improving model systems, although the stromal compartment is of murine origin and often poorly developed, and strict clinical relevance is still unclear. Patient-derived xenograft (PDX) models may provide better model systems going forward, since direct xenografts can retain some human stroma.15Yoshida G.J. Applications of patient-derived tumor xenograft models and tumor organoids.J. Hematol. Oncol. 2020; 13: 4Crossref PubMed Scopus (66) Google Scholar Many of the pharmacodynamic studies discussed below make use of human ex vivo tumor culture systems, which usually contain all of the representative stromal cell compartments but suffer from limited viability and useful lifespan.16Scott E.M. Frost S. Khalique H. Freedman J.D. Seymour L.W. Lei-Rossmann J. Use of liquid patient ascites fluids as a preclinical model for oncolytic virus activity.Methods Mol. Biol. 2020; 2058: 261-270Crossref PubMed Scopus (2) Google Scholar All of these model systems bring their own technical challenges, and we have tried to interpret findings with this in mind throughout this review. One major advantage of encoding anti-cancer biologics within gene therapy vectors or OVs is the potential for selective expression within tumors. While this does not increase the amount of material being delivered to the tumor, local production in situ leads to high local concentrations of the secreted biologic within the TME and relatively low concentrations in the circulation, thereby maximizing the therapeutic effect and limiting systemic toxicities. This strategy is most effective when the expressed agents are relatively small macromolecules, improving their percolation through the TME and also minimizing their persistence in the bloodstream (by accelerated renal clearance) if they leach out of the tumor into the circulation. For example, small biologics such as nanobodies (∼15 kDa) and single-chain variable fragment (scFv) (∼28 kDa) have a relatively short half-life of <0.5 h.17Hutt M. Färber-Schwarz A. Unverdorben F. Richter F. Kontermann R.E. Plasma half-life extension of small recombinant antibodies by fusion to immunoglobulin-binding domains.J. Biol. Chem. 2012; 287: 4462-4469Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar,18Hamers-Casterman C. Atarhouch T. Muyldermans S. Robinson G. Hamers C. Songa E.B. Bendahman N. Hamers R. Naturally occurring antibodies devoid of light chains.Nature. 1993; 363: 446-448Crossref PubMed Scopus (1878) Google Scholar This is the converse of the desirable physical properties for systemic delivery, where a long half-life (for example using an intact antibody) is normally seen as preferred to maximize the opportunity to access disseminated targets. Accordingly, from a pharmacokinetic perspective, encoding antibody fragments or small biologics within gene therapy vectors or OVs may comprise a particularly powerful approach. For gene therapies, transgene expression is normally regulated using tumor- or tissue-selective promoters.19Hermiston T.W. Kuhn I. Armed therapeutic viruses: strategies and challenges to arming oncolytic viruses with therapeutic genes.Cancer Gene Ther. 2002; 9: 1022-1035Crossref PubMed Scopus (122) Google Scholar However, OVs provide the opportunity to use endogenous viral promoters to regulate expression, linking expression to the virus life cycle and thereby exploiting the cancer selectivity of the OV to ensure cancer-selective expression of the transgene.20Robinson M. Ge Y. Ko D. Yendluri S. Laflamme G. Hawkins L. Jooss K. Comparison of the E3 and L3 regions for arming oncolytic adenoviruses to achieve a high level of tumor-specific transgene expression.Cancer Gene Ther. 2008; 15: 9-17Crossref PubMed Scopus (8) Google Scholar Alternatively, tumor- or tissue-specific promoters such as telomerase reverse transcriptase promoter21Wirth T. Zender L. Schulte B. Mundt B. Plentz R. Rudolph K.L. Manns M. Kubicka S. Kühnel F. A telomerase-dependent conditionally replicating adenovirus for selective treatment of cancer.Cancer Res. 2003; 63: 3181-3188PubMed Google Scholar and prostate-specific antigen promoter22DeWeese T.L. van der Poel H. Li S. Mikhak B. Drew R. Goemann M. Hamper U. DeJong R. Detorie N. Rodriguez R. et al.A phase I trial of CV706, a replication-competent, PSA selective oncolytic adenovirus, for the treatment of locally recurrent prostate cancer following radiation therapy.Cancer Res. 2001; 61: 7464-7472PubMed Google Scholar have been used to regulate both conditional replication and transgene expression in selected tissues. This also gives considerable flexibility over the duration of transgene expression. The use of AAV (adeno-associated virus) for gene therapy can give extended transgene expression; however, for most cancer therapies, where the expressed agents are likely to cause cytotoxicity, the intrinsically limited duration of expression afforded by most OVs may be preferable. Cancer-associated fibroblasts (CAFs) orchestrate several aspects of cancer biology that contribute to poor prognosis, often producing cytokines, chemokines, and extracellular matrix (ECM). Notably, they secrete vascular endothelial growth factor (VEGF) to drive angiogenesis and transforming growth factor (TGF)-β to polarize immune cells to become immunosuppressive.23Chen X. Song E. Turning foes to friends: Targeting cancer-associated fibroblasts.Nat. Rev. Drug Discov. 2019; 18: 99-115Crossref PubMed Scopus (353) Google Scholar In addition, CAFs can express high levels of programmed cell death ligand 2 (PD-L2) and FAS ligand (FASLG) to promote tumor immune evasion.24Lakins M.A. Ghorani E. Munir H. Martins C.P. Shields J.D. Cancer-associated fibroblasts induce antigen-specific deletion of CD8 + T cells to protect tumour cells.Nat. 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Ther. 2007; 15: 1982-1990Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar Improved viral distribution and therapeutic outcome were also observed using an oncolytic herpes simplex virus 1 (HSV-1) vector after co-injection with bacterial collagenase to degrade fibrillar collagen in a melanoma xenograft mouse model.25McKee T.D. Grandi P. Mok W. Alexandrakis G. Insin N. Zimmer J.P. Bawendi M.G. Boucher Y. Breakefield X.O. Jain R.K. Degradation of fibrillar collagen in a human melanoma xenograft improves the efficacy of an oncolytic herpes simplex virus vector.Cancer Res. 2006; 66: 2509-2513Crossref PubMed Scopus (310) Google Scholar While extracellular proteins provide the structural framework for ECM, more flexible hydrophilic components can associate with it and increase viscosity, further restricting virus spread by providing even greater resistance to fluid convection. These components include hyaluronic acid and extracellular DNA released during non-apoptotic tumor cell death. Building on observations that PEG-PH20 (hyaluronidase derivatized with poly(ethylene glycol) to improve its stability in the bloodstream) could be used to decrease the interstitial fluid pressure (IFP) and enhance the delivery of macromolecules and viruses into tumors,33Thompson C.B. Shepard H.M. O'Connor P.M. Kadhim S. Jiang P. Osgood R.J. Bookbinder L.H. Li X. Sugarman B.J. Connor R.J. et al.Enzymatic depletion of tumor hyaluronan induces antitumor responses in preclinical animal models.Mol. Cancer Ther. 2010; 9: 3052-3064Crossref PubMed Scopus (192) Google Scholar several groups have explored hyaluronidase-armed Ad or Ad co-injected with hyaluronidase to degrade the hyaluronan-rich matrix in an attempt to improve virus penetration. Guedan et al.34Guedan S. Rojas J.J. Gros A. Mercade E. Cascallo M. Alemany R. 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Tumor-stroma interactions reduce the efficacy of adenoviral therapy through the HGF-MET pathway.Cancer Sci. 2011; 102: 484-491Crossref PubMed Scopus (5) Google Scholar Intriguingly, the expression of essential Ad receptors (the coxsackievirus and Ad receptor, integrins αvβ3 and αvβ5, as well as clathrin) was found to be downregulated in pancreatic cancer cells exposed to media that had been conditioned with patient-derived fibroblasts in vitro.40Nemerow G.R. Pache L. Reddy V. Stewart P.L. Insights into adenovirus host cell interactions from structural studies.Virology. 2009; 384: 380-388Crossref PubMed Scopus (67) Google Scholar The study implicated hepatocyte growth factor (HGF) in the conditioned-causing phosphorylation of Met in the cancer cells, since inhibition of HGF/Met restored receptor expression. CAFs can also provide physical barriers to virus infection. For example, Vähä-Koskela et al.41Vähä-Koskela M.J. Kallio J.P. Jansson L.C. Heikkilä J.E. Zakhartchenko V.A. Kallajoki M.A. Kähäri V.M. Hinkkanen A.E. Oncolytic capacity of attenuated replicative Semliki Forest virus in human melanoma xenografts in severe combined immunodeficient mice.Cancer Res. 2006; 66: 7185-7194Crossref PubMed Scopus (55) Google Scholar reported that initial oncolysis by an oncolytic Semliki Forest virus (SFV) strain A7 led to the formation of CAF-encapsulated tumor nodules containing both dying and regrowing cancer cells that were resistant to subsequent virus challenge. These studies illustrate the diverse ways that CAFs can directly impact successful gene and virotherapy by preventing infection of target cells. CAFs also secrete a wide range of signaling proteins, MMPs, and angiogenic factors, all of which can contribute to tumor growth and metastasis.42Boire A. Covic L. Agarwal A. Jacques S. Sherifi S. Kuliopulos A. 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Cancer. 2020; 20: 174-186Crossref PubMed Scopus (485) Google Scholar For all of these reasons, CAFs provide an important therapeutic target, and gene therapy strategies to achieve depletion of CAFs are considered in more detail below. Although the central paradigm for OV is to achieve selective replication and lysis of cancer cells, some studies have shown the possibility to include the stromal compartment in this activity. This is attractive given the frequent association of high stromal content and poor prognosis. For example, an oncolytic Ad with Rb-binding-deleted E1A under the transcriptional control of a truncated promoter from the stroma-associated gene SPARC (secreted protein, acidic, rich in cysteine) was reported to infect and lyse both tumor and stromal cells in human ovary cancer explants and human stroma-containing xenograft tumors in mice but showed no activity in non-malignant tissue explants.46Lopez M.V. Rivera A.A. Viale D.L. Benedetti L. Cuneo N. Kimball K.J. Wang M. 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